1209 Citations
Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA Automation of the individual steps of a crystallization experiment from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments has been the response to address this issue Today large high-throughput crystallization facilities many of them open to the general user community are capable of setting up thousands of crystallization trials per day It is thus possible to test multiple constructs of each target for their ability to form crystals on a ... More
Crystallization remains the bottleneck in the crystallographic process leading from a gene to a three-dimensional model of the encoded protein or RNA. Automation of the individual steps of a crystallization experiment, from the preparation of crystallization cocktails for initial or optimization screens to the imaging of the experiments, has been the response to address this issue. Today, large high-throughput crystallization facilities, many of them open to the general user community, are capable of setting up thousands of crystallization trials per day. It is thus possible to test multiple constructs of each target for their ability to form crystals on a production-line basis. This has improved success rates and made crystallization much more convenient. High-throughput crystallization, however, cannot relieve users of the task of producing samples of high quality. Moreover, the time gained from eliminating manual preparations must now be invested in the careful evaluation of the increased number of experiments. The latter requires a sophisticated data and laboratory information-management system. A review of the current state of automation at the individual steps of crystallization with specific attention to the automation of optimization is given. Less
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane Here we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations We show that after ligand release the apo transport domain adopts a compact and occluded conformation that can traverse the membrane completing the transport cycle Sodium binding primes the transport domain to accept its substrate ... More
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues. Less
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function Consequently functions are assigned to the vast majority of proteins via automated homology-based methods with the result that as many as are incorrectly annotated or unannotated Schnoes et al PLoS Comput Biol e PMC free article PubMed Google Scholar This manuscript describes a study of the d-mannonate dehydratase ManD subgroup of the enolase superfamily ENS to investigate how function diverges as sequence diverges Previously one member of the subgroup had ... More
The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605 [PMC free article] [PubMed] [Google Scholar]). This manuscript describes a study of the d-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of d-mannonate to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized to sample sequence�function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 103 to 104 M�1 s�1) for dehydration of d-mannonate, (2) low efficiency (kcat/KM = 101 to 102 M�1 s�1) for dehydration of d-mannonate and/or d-gluconate, and 3) no-activity with either d-mannonate or d-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes d-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004, 576, 133�136 [PubMed] [Google Scholar]) (Ahmed et al. Biochem. J. 2005, 390, 529�540 [PMC free article] [PubMed] [Google Scholar]). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs. Less
The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity Despite its success the method is in its infancy as far as rational crystallogenesis is concerned Consequently significant time effort and resources are still required to generate structure-grade crystals especially with a new target type Therefore a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types Recently a strategy for crystallizing a prokaryotic a-helical membrane protein diacylglycerol kinase DgkA by the in meso method was reported Cryst Growth Des ... More
The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic a-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des. 2013, 13, 2846-2857 [PMC free article] [PubMed] [Google Scholar]). Here, we describe its application to the human a-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 �C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method. Less
PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure In cyanobacteria PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling and these interactions are modulated by ADP ATP and -oxoglutarate These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure Here we used the PII protein from Synechococcus elongatus PCC to reveal the structural basis of anti-cooperative ADP binding Furthermore we clarified the mutual influence of PII-receptor interaction and sensing of the ATP ADP ratio The crystal structures of two ... More
PII signaling proteins comprise one of the most versatile signaling devices in nature and have a highly conserved structure. In cyanobacteria, PipX and N-acetyl-l-glutamate kinase are receptors of PII signaling, and these interactions are modulated by ADP, ATP, and 2-oxoglutarate. These effector molecules bind interdependently to three anti-cooperative binding sites on the trimeric PII protein and thereby affect its structure. Here we used the PII protein from Synechococcus elongatus PCC 7942 to reveal the structural basis of anti-cooperative ADP binding. Furthermore, we clarified the mutual influence of PII-receptor interaction and sensing of the ATP/ADP ratio. The crystal structures of two forms of trimeric PII, one with one ADP bound and the other with all three ADP-binding sites occupied, revealed significant differences in the ADP binding mode: at one site (S1) ADP is tightly bound through side-chain and main-chain interactions, whereas at the other two sites (S2 and S3) the ADP molecules are only bound by main-chain interactions. In the presence of the PII-receptor PipX, the affinity of ADP to the first binding site S1 strongly increases, whereas the affinity for ATP decreases due to PipX favoring the S1 conformation of PII-ADP. In consequence, the PII-PipX interaction is highly sensitive to subtle fluctuations in the ATP/ADP ratio. By contrast, the PII-N-acetyl-l-glutamate kinase interaction, which is negatively affected by ADP, is insensitive to these fluctuations. Modulation of the metabolite-sensing properties of PII by its receptors allows PII to differentially perceive signals in a target-specific manner and to perform multitasking signal transduction. Less
Human noroviruses NoVs cause acute epidemic gastroenteritis Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens HBGAs which are also critical for NoV attachment to host cells Human NoVs are classified into two major genogroups genogroup I GI and GII with each genogroup further divided into several genotypes GII NoVs are more prevalent and exhibit periodic emergence of new variants suggested to be driven by altered HBGA binding specificities and antigenic drift Recent epidemiological studies show increased activity among GI NoVs with some members showing the ability to bind nonsecretor HBGAs ... More
Human noroviruses (NoVs) cause acute epidemic gastroenteritis. Susceptibility to the majority of NoV infections is determined by genetically controlled secretor-dependent expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. Human NoVs are classified into two major genogroups (genogroup I [GI] and GII), with each genogroup further divided into several genotypes. GII NoVs are more prevalent and exhibit periodic emergence of new variants, suggested to be driven by altered HBGA binding specificities and antigenic drift. Recent epidemiological studies show increased activity among GI NoVs, with some members showing the ability to bind nonsecretor HBGAs. NoVs bind HBGAs through the protruding (P) domain of the major capsid protein VP1. GI NoVs, similar to GII, exhibit significant sequence variations in the P domain; it is unclear how these variations affect HBGA binding specificities. To understand the determinants of possible strain-specific HBGA binding among GI NoVs, we determined the structure of the P domain of a GI.7 clinical isolate and compared it to the previously determined P domain structures of GI.1 and GI.2 strains. Our crystallographic studies revealed significant structural differences, particularly in the loop regions of the GI.7 P domain, altering its surface topography and electrostatic landscape and potentially indicating antigenic variation. The GI.7 strain bound to H- and A-type, Lewis secretor, and Lewis nonsecretor families of HBGAs, allowing us to further elucidate the structural determinants of nonsecretor HBGA binding among GI NoVs and to infer several contrasting and generalizable features of HBGA binding in the GI NoVs. Less
In the Gram-negative enterobacterium Erwinia Pectobacterium and Serratia sp ATCC intrinsic resistance to the carbapenem antibiotic -carbapen- -em- -carboxylic acid is mediated by the CarF and CarG proteins by an unknown mechanism Here we report a high-resolution crystal structure for the Serratia sp ATCC carbapenem resistance protein CarG This structure of CarG is the first in the carbapenem intrinsic resistance CIR family of resistance proteins from carbapenem-producing bacteria The crystal structure shows the protein to form a homodimer in agreement with results from analytical gel filtration The structure of CarG does not show homology with any known antibiotic resistance proteins ... More
In the Gram-negative enterobacterium Erwinia (Pectobacterium) and Serratia sp. ATCC 39006, intrinsic resistance to the carbapenem antibiotic 1-carbapen-2-em-3-carboxylic acid is mediated by the CarF and CarG proteins, by an unknown mechanism. Here, we report a high-resolution crystal structure for the Serratia sp. ATCC 39006 carbapenem resistance protein CarG. This structure of CarG is the first in the carbapenem intrinsic resistance (CIR) family of resistance proteins from carbapenem-producing bacteria. The crystal structure shows the protein to form a homodimer, in agreement with results from analytical gel filtration. The structure of CarG does not show homology with any known antibiotic resistance proteins nor does it belong to any well-characterised protein structural family. However, it is a close structural homologue of the bacterial inhibitor of invertebrate lysozyme, PliI-Ah, with some interesting structural variations, including the absence of the catalytic site responsible for lysozyme inhibition. Both proteins show a unique �-sandwich fold with short terminal a-helices. The core of the protein is formed by stacked anti-parallel sheets that are individually very similar in the two proteins but differ in their packing interface, causing the splaying of the two sheets in CarG. Furthermore, a conserved cation binding site identified in CarG is absent from the homologue. Less
The vertebrate sodium Nav channel is composed of an ion-conducting a subunit and associated subunits Here we report the crystal structure of the human subunit immunoglobulin Ig domain a functionally important component of Nav channels in neurons and cardiomyocytes Surprisingly we found that the subunit Ig domain assembles as a trimer in the crystal asymmetric unit Analytical ultracentrifugation confirmed the presence of Ig domain monomers dimers and trimers in free solution and atomic force microscopy imaging also detected full-length subunit monomers dimers and trimers Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers ... More
The vertebrate sodium (Nav) channel is composed of an ion-conducting a subunit and associated � subunits. Here, we report the crystal structure of the human �3 subunit immunoglobulin (Ig) domain, a functionally important component of Nav channels in neurons and cardiomyocytes. Surprisingly, we found that the �3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length �3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length �3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that �3 subunits can bind to more than one site on the Nav 1.5 a subunit and induce the formation of a subunit oligomers, including trimers. Our results suggest a new and unexpected role for the �3 subunits in Nav channel cross-linking and provide new structural insights into some pathological Nav channel mutations. Less
Lipidic cubic phase LCP crystallization has proven successful for high-resolution structure determination of challenging membrane proteins Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals providing a continuously renewed source of material for serial femtosecond crystallography Data collected from sub- - m-sized crystals produced with less than mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor
Antibodies m and F are the only effective human HIV- -neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region MPER of the gp subunit of the HIV- envelope glycoprotein Although F has been extensively characterized much less is known about antibody m or antibody m a closely related light-chain variant Here we report the crystal structure of m in complex with its gp epitope along with unbound structures of m and m We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp and determined the molecular basis that ... More
Antibodies m66.6 and 2F5 are the only effective human HIV-1-neutralizing antibodies reported thus far to recognize the N-terminal region of the membrane-proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein. Although 2F5 has been extensively characterized, much less is known about antibody m66.6 or antibody m66, a closely related light-chain variant. Here, we report the crystal structure of m66 in complex with its gp41 epitope, along with unbound structures of m66 and m66.6. We used mutational and binding analyses to decipher antibody elements critical for their recognition of gp41 and determined the molecular basis that underlies their neutralization of HIV-1. When bound by m66, the N-terminal region of the gp41 MPER adopts a conformation comprising a helix, followed by an extended loop. Comparison of gp41-bound m66 to unbound m66.6 identified three light-chain residues of m66.6 that were confirmed through mutagenesis to underlie the greater breadth of m66.6-mediated virus neutralization. Recognition of gp41 by m66 also revealed similarities to antibody 2F5 both in the conformation of crucial epitope residues as well as in the angle of antibody approach. Aromatic residues at the tip of the m66.6 heavy-chain third complementarity-determining region, as in the case of 2F5, were determined to be critical for virus neutralization in a manner that correlated with antibody recognition of the MPER in a lipid context. Antibodies m66, m66.6, and 2F5 thus utilize similar mechanistic elements to recognize a common gp41-MPER epitope and to neutralize HIV-1. Less
The multidrug resistance-encoding IncA C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria A plasmid-encoded disulfide isomerase is associated with conjugation Sequence analysis of several IncA C plasmids and IncA C-related integrative and conjugative elements ICE from commensal and pathogenic bacteria identified a conserved DsbC DsbG homolog DsbP The crystal structure of DsbP reveals an N-terminal domain a linker region and a C-terminal catalytic domain A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs Overall the structure and redox properties of ... More
The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer. Less
Opioids represent widely prescribed and abused medications although their signal transduction mechanisms are not well understood Here we present the high-resolution crystal structure of the human -opioid receptor -OR revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity The distinctive -OR sodium ion site architecture is centrally located in a polar interaction network in the -transmembrane bundle core with the sodium ion stabilizing a reduced agonist affinity state and thereby modulating signal transduction Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn to alanine ... More
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn131 to alanine or valine augments constitutive arrestin-ergic signaling. Asp95Ala, Asn310Ala, and Asn314Ala mutations transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealing that sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor. Less
Protein arginine methyltransferase PRMT is a unique but less characterized member of the family of protein arginine methyltransferases PRMTs that plays a role in male germline gene imprinting PRMT is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem PRMT genes from five different species were cloned and expressed in Escherichia coli and Sf insect cells Four gave soluble proteins from Sf cells of which two were homogeneous and one gave crystals The mouse PRMT structure was solved by the single anomalous dispersion method ... More
Protein arginine methyltransferase 7 (PRMT7) is a unique but less characterized member of the family of protein arginine methyltransferases (PRMTs) that plays a role in male germline gene imprinting. PRMT7 is the only known PRMT member that catalyzes the monomethylation but not the dimethylation of the target arginine residues and harbours two catalytic domains in tandem. PRMT7 genes from five different species were cloned and expressed in Escherichia coli and Sf21 insect cells. Four gave soluble proteins from Sf21 cells, of which two were homogeneous and one gave crystals. The mouse PRMT7 structure was solved by the single anomalous dispersion method using a crystal soaked with thimerosal that diffracted to beyond 2.1 � resolution. The crystal belonged to space group P43212, with unit-cell parameters a = b = 97.4, c = 168.1 � and one PRMT7 monomer in the asymmetric unit. The structure of another crystal form belonging to space group I222 was solved by molecular replacement. Less
The ryanodine receptor RyR is a heterotetrameric Ca release channel located on the sarcoplasmic reticulum SR membrane of different cell types RyR type RyR is the dominant isoform in skeletal muscle and RyR type RyR is abundant in the heart The RyR N-terminus is a large cytoplasmic domain that binds many channel modulators including the immunophilin calstabin Calstabins FKBPs which are cis-trans peptidyl-prolyl isomerases modify and bind to RyRs Calstabin FKBP is associated with RyR and calstabin FKBP binds to RyR The binding site for calstabins on RyRs has been studied and includes a proline The proline is preceded by ... More
The ryanodine receptor (RyR) is a heterotetrameric Ca2+ release channel located
on the sarcoplasmic reticulum (SR) membrane of different cell types. RyR type 1 (RyR1)
is the dominant isoform in skeletal muscle and RyR type 2 (RyR2) is abundant in the
heart. The RyR N-terminus is a large cytoplasmic domain that binds many channel
modulators, including the immunophilin calstabin.
Calstabins (FKBPs) which are cis-trans peptidyl-prolyl isomerases modify
and bind to RyRs. Calstabin1 (FKBP12) is associated with RyR1 and calstabin2
(FKBP12.6) binds to RyR2. The binding site for calstabins on RyRs has been studied
and includes a proline. The proline is preceded by a valine or an isoleucine in both RyR
isoforms. Calstabins bind to the immunosuppressive drugs rapamycin and FK506; this
binding suppresses the isomerase activity of these drugs. It has been proposed that this
inhibition is caused by the ability of the immunosuppressive compounds to mimic the
transition state of ligand isomerization.
RyR undergoes several types of post-translational modifications. One of these
modifications, phosphorylation by protein kinase A (PKA) at Ser2808, causes a decrease
in affinity of calstabin to the channel. The dissociation of calstabin from the channel
increases channel openings and promotes sub-conductance states. This phenomenon
causes Ca2+ �leak� from the SR into the cytoplasm and depletes the Ca2+ stores of the cell.
The aberrant release of Ca2+
can promote different disease states. For example, SR Ca
2+
leak in cardiac cells can promote heart failure (HF) and fatal ventricular arrhythmias.
The Marks lab demonstrated that a calstabin2 mutant � in which Asp37 was
mutated into valine � retained the ability to bind to PKA-phosphorylated channels.
Single channel measurements have shown that binding of the calstabin2-D37V restored
the calstabin2-bound channel properties.
In the present study we aimed to structurally understand the differences in binding
between wt-calstabin2 and D37V-calstabin2. To this end, we cloned, expressed and
purified the D37V-calstabin2 with an MBP fusion protein. The fusion protein was
crystallized in the presence of rapamycin and the structure was solved using molecular
replacement techniques. The main difference between the mutant and wt calstabin2 was
that a hydrogen bond between D37 and rapamycin was replaced with a van der Waals
interaction.
We also docked the mutant calstabin2-D37V into our cryo-EM structure of RyR1.
We were able to clearly see that the amino acids D (or V) interacted with a helix
projecting from the RyR structure, which we believe to contain the proline previously
identified by the Marks group. Calstabin2 interacted with the receptor via three distinct
domains; this interaction has implications for coupled gating, phosphorylation and
disease-associated mutations.
The binding affinity of the wt and mutant calstabins was measured using
radiolabeled versions of wt and D37V proteins. We found that the affinity of wt
calstabin2 to PKA-phosphorylated RyR2 decreased threefold compared to non-
phosphorylated RyR. The D37V mutant, however, was able to bind to both
phosphorylated and non-phosphorylated RyR2 with the same affinity.
This study also included efforts to crystallize different RyR fragments. We
attempted to crystallize RyR1 and RyR2 domains that are involved in RyR regulation by
small modulators or domains that are important to its activity. Despite not being able to
crystallize these fragments, we present our results here and suggest they could serve us in
the future for a variety of biochemical and biophysical studies Less
on the sarcoplasmic reticulum (SR) membrane of different cell types. RyR type 1 (RyR1)
is the dominant isoform in skeletal muscle and RyR type 2 (RyR2) is abundant in the
heart. The RyR N-terminus is a large cytoplasmic domain that binds many channel
modulators, including the immunophilin calstabin.
Calstabins (FKBPs) which are cis-trans peptidyl-prolyl isomerases modify
and bind to RyRs. Calstabin1 (FKBP12) is associated with RyR1 and calstabin2
(FKBP12.6) binds to RyR2. The binding site for calstabins on RyRs has been studied
and includes a proline. The proline is preceded by a valine or an isoleucine in both RyR
isoforms. Calstabins bind to the immunosuppressive drugs rapamycin and FK506; this
binding suppresses the isomerase activity of these drugs. It has been proposed that this
inhibition is caused by the ability of the immunosuppressive compounds to mimic the
transition state of ligand isomerization.
RyR undergoes several types of post-translational modifications. One of these
modifications, phosphorylation by protein kinase A (PKA) at Ser2808, causes a decrease
in affinity of calstabin to the channel. The dissociation of calstabin from the channel
increases channel openings and promotes sub-conductance states. This phenomenon
causes Ca2+ �leak� from the SR into the cytoplasm and depletes the Ca2+ stores of the cell.
The aberrant release of Ca2+
can promote different disease states. For example, SR Ca
2+
leak in cardiac cells can promote heart failure (HF) and fatal ventricular arrhythmias.
The Marks lab demonstrated that a calstabin2 mutant � in which Asp37 was
mutated into valine � retained the ability to bind to PKA-phosphorylated channels.
Single channel measurements have shown that binding of the calstabin2-D37V restored
the calstabin2-bound channel properties.
In the present study we aimed to structurally understand the differences in binding
between wt-calstabin2 and D37V-calstabin2. To this end, we cloned, expressed and
purified the D37V-calstabin2 with an MBP fusion protein. The fusion protein was
crystallized in the presence of rapamycin and the structure was solved using molecular
replacement techniques. The main difference between the mutant and wt calstabin2 was
that a hydrogen bond between D37 and rapamycin was replaced with a van der Waals
interaction.
We also docked the mutant calstabin2-D37V into our cryo-EM structure of RyR1.
We were able to clearly see that the amino acids D (or V) interacted with a helix
projecting from the RyR structure, which we believe to contain the proline previously
identified by the Marks group. Calstabin2 interacted with the receptor via three distinct
domains; this interaction has implications for coupled gating, phosphorylation and
disease-associated mutations.
The binding affinity of the wt and mutant calstabins was measured using
radiolabeled versions of wt and D37V proteins. We found that the affinity of wt
calstabin2 to PKA-phosphorylated RyR2 decreased threefold compared to non-
phosphorylated RyR. The D37V mutant, however, was able to bind to both
phosphorylated and non-phosphorylated RyR2 with the same affinity.
This study also included efforts to crystallize different RyR fragments. We
attempted to crystallize RyR1 and RyR2 domains that are involved in RyR regulation by
small modulators or domains that are important to its activity. Despite not being able to
crystallize these fragments, we present our results here and suggest they could serve us in
the future for a variety of biochemical and biophysical studies Less
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data In chapter several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process The new MULTICOMB program discussed in chapter provides a new phase combination algorithm for the density modification step of the structure solution process MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction SAD data directly accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment Testing of ... More
CRANK is a suite that links different macromolecular X-ray crystallographic programs to solve macromolecular crystal structures automatically from experimental phasing data. In chapter 2, several new algorithms implemented within CRANK increase the robustness and speed of the structure solution process. The new MULTICOMB program, discussed in chapter 3, provides a new phase combination algorithm for the density modification step of the structure solution process. MULTICOMB implements a novel advanced multivariate function that considers the single-wavelength anomalous diffraction (SAD) data directly, accounts for the correlation between the initial and density-modified maps and refines errors that can occur in a SAD experiment. Testing of these new algorithms with over 100 real data sets showed a dramatic improvement over state-of-the-art methods. These novel methods were also applied in solving the new structure of the DNA-binding protein Sso10a2 from Sulfolobus solfataricus reported in chapter 4. This structure provides insight to the observed differences in behaviour between Sso10a2 and its close homolog Sso10a. The last chapter of this work describes the crystallization conditions for a recombinant, fully glycosylated form of the human C1 inhibitor protein, which is involved in hereditary angioedema, a potentially life threatening condition. Less
Studies were undertaken to assess the merits and limitations of second-harmonic generation SHG for the selective detection of protein and polypeptide crystal formation focusing on the potential for false positives from SHG-active salts present in crystallization media The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples Out of salts investigated six produced significant background SHG and of the wells of a sparse-matrix screen produced SHG upon solvent evaporation SHG-active salts include phosphates hydrated sulfates formates and tartrates while chlorides acetates and anhydrous sulfates resulted in no detectable SHG activity The identified ... More
Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants. Less
Bacterial DsbA enzymes catalyze oxidative folding of virulence factors and have been identified as targets for antivirulence drugs However DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA EcDsbA Nonetheless sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA For example Salmonella enterica typhimurium SeDsbA sequence identity to EcDsbA shares almost identical structural surface and redox properties Using comparative sequence and structure analysis ... More
Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class. Less
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained This methodology is time consuming and resource intensive hindering protein structure determination Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I PSI due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements Structure determination techniques tailored for these difficult to crystallize proteins such as femtosecond nanocrystallography are being developed yet still need specific crystal characteristics Here we demonstrate a simple and robust ... More
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these �difficult to crystallize� proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. Less
The Toll interleukin- receptor TIR domain is a protein protein interaction domain that is found in both animal and plant immune receptors The N-terminal TIR domain from the nucleotide-binding NB leucine-rich repeat LRR class of plant disease-resistance R proteins has been shown to play an important role in defence signalling Recently the crystal structure of the TIR domain from flax R protein L was determined and this structure combined with functional studies demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L To advance the molecular understanding of the function of TIR domains in R-protein signalling ... More
The Toll/interleukin-1 receptor (TIR) domain is a protein–protein interaction domain that is found in both animal and plant immune receptors. The N-terminal TIR domain from the nucleotide-binding (NB)–leucine-rich repeat (LRR) class of plant disease-resistance (R) proteins has been shown to play an important role in defence signalling. Recently, the crystal structure of the TIR domain from flax R protein L6 was determined and this structure, combined with functional studies, demonstrated that TIR-domain homodimerization is a requirement for function of the R protein L6. To advance the molecular understanding of the function of TIR domains in R-protein signalling, the protein expression, purification, crystallization and X-ray diffraction analyses of the TIR domains of the Arabidopsis thaliana R proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) and the resistance-like protein SNC1 (suppressor of npr1-1, constitutive 1) are reported here. RPS4 and RRS1 function cooperatively as a dual resistance-protein system that prevents infection by three distinct pathogens. SNC1 is implicated in resistance pathways in Arabidopsis and is believed to be involved in transcriptional regulation through its interaction with the transcriptional corepressor TPR1 (Topless-related 1). The TIR domains of all three proteins have successfully been expressed and purified as soluble proteins in Escherichia coli. Plate-like crystals of the RPS4 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.05 Å resolution, had the symmetry of space group P1 and analysis of the Matthews coefficient suggested that there were four molecules per asymmetric unit. Tetragonal crystals of the RRS1 TIR domain were obtained using ammonium sulfate as a precipitant; they diffracted X-rays to 1.75 Å resolution, had the symmetry of space group P41212 or P43212 and were most likely to contain one molecule per asymmetric unit. Crystals of the SNC1 TIR domain were obtained using PEG 3350 as a precipitant; they diffracted X-rays to 2.20 Å resolution and had the symmetry of space group P41212 or P43212, with two molecules predicted per asymmetric unit. These results provide a good foundation to advance the molecular and structural understanding of the function of the TIR domain in plant innate immunity. Less
Current HIV- vaccines elicit strain-specific neutralizing antibodies However cross-reactive neutralizing antibodies arise in of HIV- -infected individuals and details of their generation could provide a roadmap for effective vaccination Here we report the isolation evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection The mature antibody CH neutralized of HIV- isolates and its co-crystal structure with gp revealed a novel loop-based mechanism of CD -binding site recognition Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation Notably the CH -lineage unmutated common ancestor avidly bound the transmitted founder HIV- ... More
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in ~20% of HIV-1-infected individuals, and details of their generation could provide a roadmap for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection. The mature antibody, CH103, neutralized ~55% of HIV-1 isolates, and its co-crystal structure with gp120 revealed a novel loop-based mechanism of CD4-binding site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the CH103-lineage unmutated common ancestor avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data elucidate the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies and provide insights into strategies to elicit similar antibodies via vaccination. Less
Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals thus overcoming the experimental riddle faced in traditional crystallography methods Here we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations Crystals ... More
Structure elucidation of large membrane protein complexes still comprises a considerable challenge yet is a key factor in drug development and disease combat. Femtosecond nanocrystallography is an emerging technique with which structural information of membrane proteins is obtained without the need to grow large crystals, thus overcoming the experimental riddle faced in traditional crystallography methods. Here, we demonstrate for the first time a microfluidic device capable of sorting membrane protein crystals based on size using dielectrophoresis. We demonstrate the excellent sorting power of this new approach with numerical simulations of selected sub-micrometer beads in excellent agreement with experimental observations. Crystals from batch crystallization broths of the huge membrane protein complex photosystem I were sorted without further treatment, resulting in a high degree of monodispersity and crystallinity in the ~ 100 nm size range. Microfluidic integration, continuous sorting, and nanometer-sized crystal fractions make this method ideal for direct coupling to femtosecond nanocrystallography. Less
Mechanosensitive channels MS are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture Therefore these channels serve as emergency valves when experiencing significant environmental stress The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein So far archeal mechanosensitive channels of small conductance have ... More
Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins. Less
Aims The prototypical protein disulfide bond Dsb formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli However genomic analysis has revealed several distinct Dsb-like systems in bacteria including the pathogen Salmonella enterica serovar Typhimurium This includes the scsABCD locus which encodes a system that has been shown via genetic analysis to confer copper tolerance but whose biochemical properties at the protein level are not defined The aim of this study was to provide functional insights into the soluble ScsC protein through structural biochemical and genetic analyses Results Here we ... More
Aims: The prototypical protein disulfide bond (Dsb) formation and protein refolding pathways in the bacterial periplasm involving Dsb proteins have been most comprehensively defined in Escherichia coli. However, genomic analysis has revealed several distinct Dsb-like systems in bacteria, including the pathogen Salmonella enterica serovar Typhimurium. This includes the scsABCD locus, which encodes a system that has been shown via genetic analysis to confer copper tolerance, but whose biochemical properties at the protein level are not defined. The aim of this study was to provide functional insights into the soluble ScsC protein through structural, biochemical, and genetic analyses. Results: Here we describe the structural and biochemical characterization of ScsC, the soluble DsbA-like component of this system. Our crystal structure of ScsC reveals a similar overall fold to DsbA, although the topology of �-sheets and a-helices in the thioredoxin domains differ. The midpoint reduction potential of the CXXC active site in ScsC was determined to be -132 mV versus normal hydrogen electrode. The reactive site cysteine has a low pKa, typical of the nucleophilic cysteines found in DsbA-like proteins. Deletion of scsC from S. Typhimurium elicits sensitivity to copper (II) ions, suggesting a potential involvement for ScsC in disulfide folding under conditions of copper stress. Innovation and Conclusion: ScsC is a novel disulfide oxidoreductase involved in protection against copper ion toxicity. Antioxid. Redox Signal. 19, 1494�1506. Less
In mammals Toll-like receptors TLRs recognize conserved microbial molecular signatures and induce an early innate immune response in the host TLR signalling is mediated by interactions between the cytosolic TIR Toll interleukin- receptor domains of the receptor and the adaptor proteins Increasingly it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis Studies have shown that TcpB interferes with the TLR and TLR signalling pathways to inhibit TLR-mediated inflammatory responses Such interference may involve TIR TIR-domain interactions between bacterial and mammalian ... More
In mammals, Toll-like receptors (TLRs) recognize conserved microbial molecular signatures and induce an early innate immune response in the host. TLR signalling is mediated by interactions between the cytosolic TIR (Toll/interleukin-1 receptor) domains of the receptor and the adaptor proteins. Increasingly, it is apparent that pathogens target this interaction via pathogen-expressed TIR-domain-containing proteins to modulate immune responses. A TIR-domain-containing protein TcpB has been reported in the pathogenic bacterium Brucella melitensis. Studies have shown that TcpB interferes with the TLR2 and TLR4 signalling pathways to inhibit TLR-mediated inflammatory responses. Such interference may involve TIR�TIR-domain interactions between bacterial and mammalian proteins, but there is a lack of information about these interactions at the molecular level. In this study, the cloning, expression, purification, crystallization and preliminary X-ray crystallographic analysis of the protein construct corresponding to the TIR domain of TcpB (residues 120�250) are reported. The crystals diffracted to 2.6 � resolution, have the symmetry of the monoclinic space group P21 and are most likely to contain four molecules in the asymmetric unit. The structure should help in understanding the molecular basis of how TcpB affects the innate immunity of the host. Less
The plant hormones cytokinins play a central role in regulating cell division and developmental events Cytokinin oxidase regulates the levels of these plant hormones by catalyzing their irreversible oxidation which contributes to the regulation of various morpho-physiological processes controlled by cytokinins In this study the crystallization and preliminary X-ray diffraction analysis of the flax cytokinin oxidase LuCKX are reported Plate-like crystals of LuCKX were obtained using PEG as a precipitant and diffracted X-rays to resolution The protein crystals have the symmetry of space group C and are most likely to contain two molecules per asymmetric unit
The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors The archetypal disulfide oxidase proteins in Escherichia coli Ec are DsbA and DsbB which together form a functional unit DsbA introduces disulfides into folding proteins and DsbB re oxidizes DsbA to maintain it in the active form In Mycobacterium tuberculosis Mtb no DsbB homologue is encoded but a functionally similar but structurally divergent protein MtbVKOR has been identified Here the Mtb protein Rv c is investigated and it is shown that it is the DsbA-like partner ... More
The bacterial disulfide machinery is an attractive molecular target for developing new antibacterials because it is required for the production of multiple virulence factors. The archetypal disulfide oxidase proteins in Escherichia coli (Ec) are DsbA and DsbB, which together form a functional unit: DsbA introduces disulfides into folding proteins and DsbB re�oxidizes DsbA to maintain it in the active form. In Mycobacterium tuberculosis (Mtb), no DsbB homologue is encoded but a functionally similar but structurally divergent protein, MtbVKOR, has been identified. Here, the Mtb protein Rv2969c is investigated and it is shown that it is the DsbA-like partner protein of MtbVKOR. It is found that it has the characteristic redox features of a DsbA-like protein: a highly acidic catalytic cysteine, a highly oxidizing potential and a destabilizing active-site disulfide bond. Rv2969c also has peptide-oxidizing activity and recognizes peptide segments derived from the periplasmic loops of MtbVKOR. Unlike the archetypal EcDsbA enzyme, Rv2969c has little or no activity in disulfide-reducing and disulfide-isomerase assays. The crystal structure of Rv2969c reveals a canonical DsbA fold comprising a thioredoxin domain with an embedded helical domain. However, Rv2969c diverges considerably from other DsbAs, including having an additional C-terminal helix (H8) that may restrain the mobility of the catalytic helix H1. The enzyme is also characterized by a very shallow hydrophobic binding surface and a negative electrostatic surface potential surrounding the catalytic cysteine. The structure of Rv2969c was also used to model the structure of a paralogous DsbA-like domain of the Ser/Thr protein kinase PknE. Together, these results show that Rv2969c is a DsbA-like protein with unique properties and a limited substrate-binding specificity. Less
Nuclear pore complexes NPCs are giant molecular machines embedded in the double membrane that surrounds the eukaryotic nucleus Deciphering the molecular structure of the NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport In less than a decade atomic level structures of many nucleoporins Nups have been solved and the molecular picture of the NPC is becoming increasingly clearer Nup subcomplex is a nucleoporin subcomplex in the NPC s central channel the molecular structure of which is not known so far At a sequence level the N-terminal half of all subunits in the ... More
Nuclear pore complexes (NPCs) are giant molecular machines embedded in the double
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold. Less
membrane that surrounds the eukaryotic nucleus. Deciphering the molecular structure of the
NPCs is critical to our understanding of both cellular architecture and the mechanism of nucleocytoplasmic transport. In less than a decade, atomic level structures of many nucleoporins
(Nups) have been solved and the molecular picture of the NPC is becoming increasingly clearer.
Nup62�58�54 subcomplex is a nucleoporin subcomplex in the NPC�s central channel, the
molecular structure of which, is not known so far. At a sequence level, the N-terminal half of all
subunits in the complex contain intrinsically disordered phenylalanine-glycine (FG) repeatmotifs. The C-terminal half is structured into coiled-coil domains that engage in tight proteinprotein interactions to hold the complex together and to anchor it to the NPC scaffold. In this
project, I aimed to elucidate the molecular structure of the X.laevis ?FG-Nup62�58�54 complex
by X-ray crystallography. Poor solubility of some coiled-coil domain containing Nups, such as
Nup54, in bacterial expression has been a challenge towards obtaining large amounts of
nucleoporins required for crystallographic analysis. We established a system where all three
subunits of xl?FGNup62�58�54 were co-expressed together in E.coli. The soluble amount of
Nup54 was greatly enhanced, when co-expressed together with its interaction partners.
Biochemical analysis of the recombinant ?FG-Nup62�58�54 complex revealed a monomeric, nonspherical complex with a clear 1:1:1 subunit stoichiometry, which had been unclear so far. Also,
in contrast to previously published reports, the ?FG-Nup62�58�54 complex did not show any
tendency to form protein-concentration dependent higher-order oligomers. However, extensive
crystallization trials failed to crystallize ?FG-Nup62�58�54 complex, probably due to inherent
flexibility in its structure. Therefore, we generated and screened a series of single-domain
antibodies (nanobodies, Nbs) against ?FG-Nup62�58�54 complex and identified a few that
stabilized the trimeric complex but did not recognize any of the individual subunits or dimeric
interactions. Crystallization trials of ?FG-xlNup62�58�54 complex conjugated to one such
nanobody, Nb15, yielded rod-shaped crystals, which are currently limited in their diffraction
potential to ~7.5�. Thus, nanobody conjugation of the ?FG-xlNup62�58�54 complex aided in its
crystallization and can be used as a general approach to stabilize NPC subcomplexes.
Furthermore, a coiled-coil interaction between xlNup93 N-terminus and xlNup62�58�54 complex
is thought to recruit the Nup62�58�54 complex to the NPCs. This interaction in our experiments,
however, turned out to be surprisingly weak. This now predicts additional interaction sites for a
high avidity anchorage of the Nup62�58�54 complex to the NPC scaffold. Less
With increasingly large immunocompromised populations around the world opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality To combat the paucity of antifungal compounds new drug targets must be investigated Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate Although the enzyme is ubiquitous and well characterized in other kingdoms no crystallographic studies on the fungal protein have been performed Presented here are the expression purification crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase The crystals had the symmetry ... More
With increasingly large immunocompromised populations around the world, opportunistic fungal pathogens such as Cryptococcus neoformans are a growing cause of morbidity and mortality. To combat the paucity of antifungal compounds, new drug targets must be investigated. Adenylosuccinate synthetase is a crucial enzyme in the ATP de novo biosynthetic pathway, catalyzing the formation of adenylosuccinate from inosine monophosphate and aspartate. Although the enzyme is ubiquitous and well characterized in other kingdoms, no crystallographic studies on the fungal protein have been performed. Presented here are the expression, purification, crystallization and initial crystallographic analyses of cryptococcal adenylosuccinate synthetase. The crystals had the symmetry of space group P212121 and diffracted to 2.2 � resolution. Less
In higher plants the MADS-box genes encode a large family of transcription factors TFs involved in key developmental processes most notably plant reproduction flowering and floral organ development SEPALLATA SEP is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs SEP is divided into four domains the M MADS domain involved in DNA binding and dimerization the I intervening domain a short domain involved in dimerization the K keratin-like domain important for multimeric MADS complex formation and the C C-terminal domain ... More
In higher plants, the MADS-box genes encode a large family of transcription factors (TFs) involved in key developmental processes, most notably plant reproduction, flowering and floral organ development. SEPALLATA 3 (SEP3) is a member of the MADS TF family and plays a role in the development of the floral organs through the formation of multiprotein complexes with other MADS-family TFs. SEP3 is divided into four domains: the M (MADS) domain, involved in DNA binding and dimerization, the I (intervening) domain, a short domain involved in dimerization, the K (keratin-like) domain important for multimeric MADS complex formation and the C (C-terminal) domain, a largely unstructured region putatively important for higher-order complex formation. The entire K domain along with a portion of the I and C domains of SEP3 was crystallized using high-throughput robotic screening followed by optimization. The crystals belonged to space group P21212, with unit-cell parameters a = 123.44, b = 143.07, c = 49.83 �, and a complete data set was collected to 2.53 � resolution. Less
During the past year electron crystallography of membrane proteins has provided structural insights into the mechanism of several different transporters and into their interactions with lipid molecules within the bilayer From a technical perspective there have been important advances in high-throughput screening of crystallization trials and in automated imaging of membrane crystals with the electron microscope There have also been key developments in software and in molecular replacement and phase extension methods designed to facilitate the process of structure determination
Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals In this context the present study reports the isolation recombinant expression biochemical and structural characterization of a novel endoxylanase family GH SCXyl identified from sugarcane soil metagenome The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH and C The crystal structure was solved at resolution revealing the classical a -barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp -Arg loop that can adopt distinct conformational states ... More
Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45�C. The crystal structure was solved at 2.75 � resolution, revealing the classical (�/a)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. Less
Nonlinear optical NLO instrumentation has been integrated with synchrotron X-ray diffraction XRD for combined single-platform analysis initially targeting applications for automated crystal centering Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning Two optical designs were constructed and characterized one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer Both instruments enabled protein crystal identification with integration times between and s per pixel representing a -fold reduction in the per-pixel exposure time relative to X-ray raster scanning Quantitative centering and analysis of phenylalanine hydroxylase ... More
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. Less
As part of the mammalian innate immune response Toll-like receptors and can signal via the adaptor protein TRIF TICAM- to elicit the production of type-I interferons and cytokines Recent studies have suggested an auto-inhibitory role for the N-terminal domain NTD of TRIF This domain has no significant sequence similarity to proteins of known structure In this paper the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA M L M are reported Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol as precipitant diffracted X-rays to resolution To facilitate phase determination two additional methionines were incorporated ... More
As part of the mammalian innate immune response, Toll-like receptors 3 and 4 can signal via the adaptor protein TRIF/TICAM-1 to elicit the production of type-I interferons and cytokines. Recent studies have suggested an auto-inhibitory role for the N-terminal domain (NTD) of TRIF. This domain has no significant sequence similarity to proteins of known structure. In this paper, the crystallization and X-ray diffraction analysis of TRIF-NTD and its selenomethionine-labelled mutant TRIF-NTDA66M/L113M are reported. Thin plate-like crystals of native TRIF-NTD obtained using polyethylene glycol 3350 as precipitant diffracted X-rays to 1.9 � resolution. To facilitate phase determination, two additional methionines were incorporated into the protein at positions chosen based on the occurrence of methionines in TRIF homologues in different species. Crystals of the selenomethionine-labelled protein were obtained under conditions similar to the wild-type protein; these crystals diffracted X-rays to 2.5 � resolution. The TRIF-NTD and TRIF-NTDA66M/L113M crystals have the symmetry of space groups P212121 and P1, and most likely contain two and four molecules in the asymmetric unit, respectively. These results provide a sound foundation for the future structure determination of this novel domain. Less
Autophagy is a degradation pathway conserved in eukaryotes Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content After fusion of the membrane an autophagosome vesicle is formed which then fuses with the vacuole or lysosome where its content is degraded PROPPINs -propeller proteins that bind polyphosphoinositides play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif The three yeast PROPPINs Atg Atg and Hsv are involved in different subtypes of autophagy In this study I purified different Atg Atg and Hsv PROPPIN homologs and showed that they bind ... More
Autophagy is a degradation pathway conserved in eukaryotes. Upon induction of autophagy a double layered membrane is formed de novo and engulfs the cytosolic content. After fusion of the membrane, an autophagosome vesicle is formed, which then fuses with the vacuole (or lysosome) where its content is degraded. PROPPINs, �-propeller proteins that bind polyphosphoinositides, play a role in autophagy and phosphoinositide binding depends on a conserved FRRG motif. The three yeast PROPPINs Atg18, Atg21 and Hsv2 are involved in different subtypes of autophagy.
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields Less
In this study, I purified different Atg18, Atg21 and Hsv2 PROPPIN homologs and showed that they bind specifically to PI3P and PI(3,5)P2 using protein-liposome co-flotation assays. Recently, we published the first structure of the PROPPIN Hsv2. Based on our structure I performed mutagenesis studies to probe phosphoinositide binding of Hsv2. I analyzed phosphoinositide binding of the alaninine mutants with liposome flotation assays. I identified conserved residues essential for binding right and left of the FRRG motif, indicating the presence of two phosphoinositide binding sites, which was an unexpected finding. Using ITC measurements I then confirmed the binding stoichiometry of two phosphoinositides to one Hsv2 molecule and determined the binding affinities of PROPPINs to both PI3P and PI(3,5)P2 incorporated in small unilamellar vesicles. Phosphoinositide binding of S. cerevisiae Hsv2 is pH dependent. Acidic environment increases and basic environment decreases the affinity. In addition, I showed the involvement of loop 6CD in membrane binding. Mutagenesis analysis of loop 6CD residues revealed that membrane insertion is dependent on both ionic and hydrophobic interactions.
Two ubiquitin-like conjugation systems modifying Atg8 (in mammals MAP1LC3) and Atg12 are essential for autophagy. Homologs of the canonical ubiquitin conjugation system, E1- and E2-like enzymes, are involved in the conjugation of Atg8 and Atg12 to their specific targets phosphatidylethanolamine and Atg5, respectively.
A in vivo reconstitution system for the two human ubiquitin-like conjugation systems Atg12 and MAP1LC3 was established using the MultiBac baculovirus expression system in insect cells. This allowed full length expression of the involved proteins and purification of the Atg5-Atg12 conjugate and lipidated MAP1LC3 in small yields Less
Structural studies of integral membrane proteins IMPs are often hampered by difficulties in producing stable homogenous samples for crystallization To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization For such an approach to be effective an efficient screening strategy is imperative To this end strategies have been developed that involve the use of green fluorescent protein GFP fusion constructs However these approaches suffer from two drawbacks proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require ... More
Structural studies of integral membrane proteins (IMPs) are often hampered by difficulties in producing stable homogenous samples for crystallization. To overcome this hurdle it has become common practice to screen large numbers of target proteins to find suitable candidates for crystallization. For such an approach to be effective, an efficient screening strategy is imperative. To this end, strategies have been developed that involve the use of green fluorescent protein (GFP) fusion constructs. However, these approaches suffer from two drawbacks; proteins with a translocated C-terminus cannot be tested and scale-up from analytical to preparative purification is often non-trivial and may require re-cloning. Less
Introduction X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution It provides key information for the understanding of protein function opening opportunities for the modulation of enzymatic mechanisms and protein ligand interactions As a consequence macromolecular crystallography plays an essential role in drug design as well as in the a posteriori validation of drug mechanisms Areas covered The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past years As a consequence access to the facilities required for these investigations such as synchrotron beamlines became more difficult and significant efforts ... More
Introduction: X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein�ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms.
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
The smoothened SMO receptor a key signal transducer in the Hedgehog Hh signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis The SMO receptor is classified as a class Frizzled class F G protein-coupled receptor GPCR although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than Here we report the crystal structure at resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY Although the SMO receptor shares the seven transmembrane helical TM fold ... More
The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. Less
The potential of second-harmonic generation SHG microscopy for automated crystal centering to guide synchrotron X- ray diffraction of protein crystals was explored These studies included i comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and ii X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging In studies using adrenergic receptor membrane-protein crystals prepared in lipidic mesophase the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X- ray minibeam ... More
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-�ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using �2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-�ray minibeam. SHG imaging was found to provide about 2 �m spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed. Less
Dynamin -like protein DNM L mediates fission of mitochondria and peroxisomes and dysfunction of DNM L has been implicated in several neurological disorders To study the molecular basis of mitochondrial remodelling we determined the crystal structure of DNM L that is comprised of a G domain a bundle signalling element and a stalk DNM L assembled via a central stalk interface and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM L on membranes and its ... More
Dynamin 1-like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria. Less
When protein crystals are submicrometre-sized X-ray radiation damage precludes conventional diffraction data collection For crystals that are of the order of nm in size at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible irrespective of the diffraction technique used Here it is shown that at a very low electron dose at most e- - a Medipix quantum area detector is sufficiently sensitive to allow the collection of a -frame rotation series of keV electron-diffraction data from a single nm thick protein crystal A highly parallel keV electron beam lambda allowed observation of ... More
When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e- �-2), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electron-diffraction data from a single ~100 nm thick protein crystal. A highly parallel 200 keV electron beam ([lambda] = 0.025 �) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4�. This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins Less
DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate In eukaryotes DING proteins have been isolated by virtue of their implication in several diseases and biological processes Some of them are potent inhibitors of HIV- replication transcription raising the question of their potential involvement in the human defence system Recently a protein from Pseudomonas aeruginosa strain PA named PA DING or LapC belonging to the DING family has been identified ... More
DING proteins form an emergent family of proteins consisting of an increasing number of homologues that have been identified in all kingdoms of life. They belong to the superfamily of phosphate-binding proteins and exhibit a high affinity for phosphate. In eukaryotes, DING proteins have been isolated by virtue of their implication in several diseases and biological processes. Some of them are potent inhibitors of HIV-1 replication/transcription, raising the question of their potential involvement in the human defence system. Recently, a protein from Pseudomonas aeruginosa strain PA14, named PA14DING or LapC, belonging to the DING family has been identified. The structure of PA14DING, combined with detailed biochemical characterization and comparative analysis with available DING protein structures, will be helpful in understanding the structural determinants implicated in the inhibition of HIV-�1 by DING proteins. Here, the expression, purification and crystallization of PA14DING and the collection of X-ray data to 1.9 � resolution are reported. Less
In hemophilia A routine prophylaxis with exogenous factor VIII FVIII requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies FVIII inhibitors To overcome these drawbacks we screened asymmetric bispecific IgG antibodies to factor IXa FIXa and factor X FX mimicking the FVIII cofactor function Since the therapeutic potential of the lead bispecific antibody was marginal FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX and the pharmacokinetics was improved by engineering the charge properties of the variable region Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain ... More
In hemophilia A, routine prophylaxis with exogenous factor VIII (FVIII) requires frequent intravenous injections and can lead to the development of anti-FVIII alloantibodies (FVIII inhibitors). To overcome these drawbacks, we screened asymmetric bispecific IgG antibodies to factor IXa (FIXa) and factor X (FX), mimicking the FVIII cofactor function. Since the therapeutic potential of the lead bispecific antibody was marginal, FVIII-mimetic activity was improved by modifying its binding properties to FIXa and FX, and the pharmacokinetics was improved by engineering the charge properties of the variable region. Difficulties in manufacturing the bispecific antibody were overcome by identifying a common light chain for the anti-FIXa and anti-FX heavy chains through framework/complementarity determining region shuffling, and by pI engineering of the two heavy chains to facilitate ion exchange chromatographic purification of the bispecific antibody from the mixture of byproducts. Engineering to overcome low solubility and deamidation was also performed. The multidimensionally optimized bispecific antibody hBS910 exhibited potent FVIII-mimetic activity in human FVIII-deficient plasma, and had a half-life of 3 weeks and high subcutaneous bioavailability in cynomolgus monkeys. Importantly, the activity of hBS910 was not affected by FVIII inhibitors, while anti-hBS910 antibodies did not inhibit FVIII activity, allowing the use of hBS910 without considering the development or presence of FVIII inhibitors. Furthermore, hBS910 could be purified on a large manufacturing scale and formulated into a subcutaneously injectable liquid formulation for clinical use. These features of hBS910 enable routine prophylaxis by subcutaneous delivery at a long dosing interval without considering the development or presence of FVIII inhibitors. We expect that hBS910 (investigational drug name: ACE910) will provide significant benefit for severe hemophilia A patients. Less
Collapsin response mediator protein- CRMP- is the latest identified member of the CRMP cytosolic phosphoprotein family which is crucial for neuronal development and repair CRMPs exist as homo- and or hetero-tetramers in vivo and participate in signaling transduction cytoskeleton rearrangements and endocytosis CRMP- antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners We determined the crystal structures of a full length and a truncated version of human CRMP- both of which form a homo-tetramer similar to those observed in CRMP- and CRMP- However solution studies indicate that CRMP- and ... More
Collapsin response mediator protein-5 (CRMP-5) is the latest identified member of the CRMP cytosolic phosphoprotein family, which is crucial for neuronal development and repair. CRMPs exist as homo- and/or hetero-tetramers�in vivo�and participate in signaling transduction, cytoskeleton rearrangements, and endocytosis. CRMP-5 antagonizes many of the other CRMPs' functions either by directly interacting with them or by competing for their binding partners. We determined the crystal structures of a full length and a truncated version of human CRMP-5, both of which form a homo-tetramer similar to those observed in CRMP-1 and CRMP-2. However, solution studies indicate that CRMP-5 and CRMP-1 form weaker homo-tetramers compared with CRMP-2, and that divalent cations, Ca2+�and Mg2+, destabilize oligomers of CRMP-5 and CRMP-1, but promote CRMP-2 oligomerization. On the basis of comparative analysis of the CRMP-5 crystal structure, we identified residues that are crucial for determining the preference for hetero-oligomer or homo-oligomer formation. We also show that in spite of being the CRMP family member most closely related to dihydropyrimidinase, CRMP-5 does not have any detectable amidohydrolase activity. The presented findings provide new detailed insights into the structure, oligomerization, and regulation of CRMPs. Less
Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of nm were imaged by kV cryo-EM on a Falcon direct electron detector The images were taken close to focus and to the eye appeared devoid of contrast Fourier transforms of the images revealed the reciprocal lattice up to resolution in favourable cases and up to resolution for about half the crystals The reciprocal-lattice spots showed structure indicating that the ordering of the crystals was not uniform Data processing revealed details at higher than resolution and indicated the presence of multiple mosaic blocks within the crystal ... More
Flash-cooled three-dimensional crystals of the small protein lysozyme with a thickness of the order of 100 nm were imaged by 300 kV cryo-EM on a Falcon direct electron detector. The images were taken close to focus and to the eye appeared devoid of contrast. Fourier transforms of the images revealed the reciprocal lattice up to 3 � resolution in favourable cases and up to 4 � resolution for about half the crystals. The reciprocal-lattice spots showed structure, indicating that the ordering of the crystals was not uniform. Data processing revealed details at higher than 2 � resolution and indicated the presence of multiple mosaic blocks within the crystal which could be separately processed. The prospects for full three-dimensional structure determination by electron imaging of protein three-dimensional nanocrystals are discussed. Less
Flavodoxins which exist widely in microorganisms have been found in various pathways with multiple physiological functions The flavodoxin Fld containing the cofactor flavin mononucleotide FMN from sulfur-reducing bacteria Desulfovibrio gigas D gigas is a short-chain enzyme that comprises residues with a molecular mass of kDa and plays important roles in the electron-transfer chain To investigate its structure we purified this Fld directly from anaerobically grown D gigas cells The crystal structure of Fld determined at resolution is a dimer with two FMN packing in an orientation head to head at a distance of which generates a long and connected negatively ... More
Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners. Less
Crystallization of membrane proteins and peptides represents a challenge in the field of structural biology Lipidic cubic phase LCP has become an important medium for crystallogenesis of membrane proteins of different molecular weight Here the small membrane peptide gramicidin is used as an example peptide to test if LCP can produce diffraction quality crystals for membrane proteins and peptides in the lower molecular weight range This approach was initially tested with the standard LCP lipid monoolein and later on extended to a variety of different monoacylglycerols varying in their acyl chain length Data sets for three different crystal forms were ... More
Crystallization of membrane proteins and peptides represents a challenge in the field of structural biology. Lipidic cubic phase (LCP) has become an important medium for crystallogenesis of membrane proteins of different molecular weight. Here, the small membrane peptide gramicidin is used as an example peptide to test if LCP can produce diffraction quality crystals for membrane proteins and peptides in the lower molecular weight range. This approach was initially tested with the standard LCP lipid monoolein and later on extended to a variety of different monoacylglycerols varying in their acyl chain length. Data sets for three different crystal forms were obtained. In each case gramicidin was found in the double stranded double helical (DSDH) conformation. One crystal form shows stabilizing hydrogen bonds between adjacent tryptophan residues indicating how DSDH can be stabilized as an aggregate in the membrane. The cytoplasmic domain of the putative zinc transporter CzrB was solved in the apo and zinc-bound forms. NMR, X-ray scattering, and size-exclusion chromatography provide support for dimer formation. Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. Less
The physical properties of viral capsids are major determinants of vaccine efficacy for several picornaviruses which impact on human and animal health Current picornavirus vaccines are frequently produced from inactivated virus Inactivation often reduces the stability of the virus capsid causing a problem for Foot and Mouth Disease Virus FMDV where certain serotypes fall apart into pentameric assemblies below pH or at temperatures slightly above C destroying their effectiveness in eliciting a protective immune response As a result vaccines require a cold chain for storage and animals need to be frequently immunised FMDV is a member of the Aphthovirus genus ... More
The physical properties of viral capsids are major determinants of vaccine efficacy for several
picornaviruses which impact on human and animal health. Current picornavirus vaccines are
frequently produced from inactivated virus. Inactivation often reduces the stability of the
virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain
serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly
above 37�C, destroying their effectiveness in eliciting a protective immune response. As a
result, vaccines require a cold chain for storage and animals need to be frequently immunised.
FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven
FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of
antigenic variation. As part of collaboration between the Division of Structural Biology,
Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South
Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious
copy virus or recombinant empty capsids with improved thermo-stability for improved
vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular
modelling, free energy calculations, X-ray crystallography, electron microscopy and various
biochemical/biophysical techniques were used to design and help characterise the capsids.
For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were
characterised by MD. Promising candidates were then engineered and shown to confer
increased thermo- and pH-stability. Thus, in silico predictions translate into marked
stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method
was used to determine crystal structures for quality assessment and to verify that no
unanticipated structural changes have occurred as a consequence of the modifications made.
The structures of the wildtype and two of the stabilised mutants were solved and the antigenic
surfaces shown to be unchanged.
Animal trials showed stabilised particles can generate a similar or improved neutralising
antibody response compared to the traditional vaccines and may therefore lead to a new
generation of stable and safe vaccines.
Declaration
ii
DECLARATION OF W Less
picornaviruses which impact on human and animal health. Current picornavirus vaccines are
frequently produced from inactivated virus. Inactivation often reduces the stability of the
virus capsid, causing a problem for Foot and Mouth Disease Virus (FMDV) where certain
serotypes fall apart into pentameric assemblies below pH 6.5 or at temperatures slightly
above 37�C, destroying their effectiveness in eliciting a protective immune response. As a
result, vaccines require a cold chain for storage and animals need to be frequently immunised.
FMDV is a member of the Aphthovirus genus of the Picornaviridae. Globally there are seven
FMDV serotypes: O, A, Asia1, C and SAT-1, -2 and -3, contributing to a dynamic pool of
antigenic variation. As part of collaboration between the Division of Structural Biology,
Oxford University, The Pirbright Institute, Reading University and ARC, Ondespoort, South
Africa we sought to rationally engineer thermo-stable FMDV capsids either as infectious
copy virus or recombinant empty capsids with improved thermo-stability for improved
vaccines. In this project, in silico molecular dynamics (MD) simulations, molecular
modelling, free energy calculations, X-ray crystallography, electron microscopy and various
biochemical/biophysical techniques were used to design and help characterise the capsids.
For the most unstable FMDV serotypes (O and SAT2), panels of stabilising mutants were
characterised by MD. Promising candidates were then engineered and shown to confer
increased thermo- and pH-stability. Thus, in silico predictions translate into marked
stabilisation of both infectious and recombinant empty viral capsids. A novel in situ method
was used to determine crystal structures for quality assessment and to verify that no
unanticipated structural changes have occurred as a consequence of the modifications made.
The structures of the wildtype and two of the stabilised mutants were solved and the antigenic
surfaces shown to be unchanged.
Animal trials showed stabilised particles can generate a similar or improved neutralising
antibody response compared to the traditional vaccines and may therefore lead to a new
generation of stable and safe vaccines.
Declaration
ii
DECLARATION OF W Less
The optimum conditions for the formation of plate-like and urchin-like microcrystals of biomolecules and their transfer to rotors for solid-state NMR spectroscopy depend on a variety of factors of which minimizing the manipulation of the microcrystals and storing the sample for several months at K C play an important role Three biological systems were investigated Hen Egg-White HEW lysozyme residues the lengthened C-terminal domain LCter of Human centrin residues and the complex between the C-terminal domain Cter of Human centrin residues and the P -XPC peptide residues
A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described It provides crystals that appear within - min of setup at K The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase which can be daunting as a material when first encountered Having used the protocol to produce crystals of the test protein lysozyme the experimenter can proceed with confidence to apply the method to more valuable membrane and soluble protein targets The ... More
A simple and inexpensive protocol for producing crystals in the sticky and viscous mesophase used for membrane protein crystallization by the in meso method is described. It provides crystals that appear within 15-30 min of setup at 293 K. The protocol gives the experimenter a convenient way of gaining familiarity and a level of comfort with the lipidic cubic mesophase, which can be daunting as a material when first encountered. Having used the protocol to produce crystals of the test protein, lysozyme, the experimenter can proceed with confidence to apply the method to more valuable membrane (and soluble) protein targets. The glass sandwich plates prepared using this robust protocol can further be used to practice harvesting and snap-cooling of in meso-grown crystals, to explore diffraction data collection with mesophase-embedded crystals, and for an assortment of quality control and calibration applications when used in combination with a crystallization robot. Less
The structure of ribose -phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC has been determined at resolution The structure was solved by molecular replacement which identified the functional homodimer in the asymmetric unit Despite only showing sequence identity to its closest homologue the structure adopted the typical a and d- ribose - phosphate isomerase fold Comparison to other related structures revealed high homology in the active site allowing a model of the substrate-bound protein to be proposed The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I - at Diamond Light ... More
The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 � resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical a and � d-�ribose 5-�phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography. Less