419 Citations
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
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
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
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
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
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
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
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
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
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
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
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
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
Including more than members in humans Rab proteins constitute the largest subfamily of the Ras- like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound inactive and a GTP-bound active conformation The conversion between these states is carried out by regulatory factors GTPase activating proteins GAPs stimulate GTP hydrolysis and guanine nucleotide exchange factors GEFs catalyze the GDP-GTP exchange Rab proteins interact with effector proteins only in the active state thereby regulating vesicular trafficking in eukaryotic cells For this purpose the activity and the intracellular localization of Rab proteins need to be tightly regulated ... More
Including more than 60 members in humans, Rab proteins constitute the largest subfamily of the Ras-
like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound
(inactive) and a GTP-bound (active) conformation. The conversion between these states is carried out by
regulatory factors: GTPase activating proteins (GAPs) stimulate GTP hydrolysis and guanine nucleotide
exchange factors (GEFs) catalyze the GDP-GTP exchange. Rab proteins interact with effector proteins
only in the active state, thereby regulating vesicular trafficking in eukaryotic cells. For this purpose, the
activity and the intracellular localization of Rab proteins need to be tightly regulated. In order to ensure
their own survival, some intracellular pathogens have developed intriguing strategies for manipulation
of intracellular vesicular transport processes and in particular of the Rab proteins involved. A prominent
example of an intracellular pathogen that manipulates Rab proteins for its own benefit is Legionella
pneumophila. In particular, the Legionella protein DrrA (defect in Rab recruitment A) was identified in
the recent past as a protein that manipulates the intracellular localization and activity of Rab1. At the
beginning of this work, structural studies on this protein showed the presence of an additional,
previously uncharacterized domain possessing adenylyltransferase activity towards Rab1. The
characterization of this enzymatic activity was the central subject of this work.
Within this work, the x-ray crystal structure of adenylylated Rab1 was solved. This structure showed that
Rab1 was specifically modified on a tyrosine residue in the functionally important switch II region.
Further studies of the effects of this modification showed that the interaction of Rab1-AMP with GAPs
and the human effector Mical-3 are drastically inhibited, whereas the interaction with the GEF domain
of the Legionella protein DrrA and the Legionella effector protein LidA are not significantly inhibited.
Characterisation of the enzyme kinetics of DrrA and the recently identified deadenylylating enzyme SidD
showed that Rab1:GTP is the preferred substrate of adenylylation by DrrA while SidD possesses a
significantly lower substrate specificity towards the active or inactive conformation of Rab1. This work
includes the first description and characterization of adenylylation as a posttranslational modification of
Rab proteins. In the context of the current literature, the results of this work allowed the proposal of a
model in which adenylylation temporarily inhibits deactivation of Rab1 by GAPs and thus the extraction
of Rab1 from the Legionella containing vacuolar (LCV) membrane by GDI and Rab1 is entrapped at the
LCV membrane. At a later stage of infection, deadenylylation by SidD allows for deactivation and
extraction by GDI. Less
like superfamily of small GTPases which act as molecular switches and can exist in a GDP-bound
(inactive) and a GTP-bound (active) conformation. The conversion between these states is carried out by
regulatory factors: GTPase activating proteins (GAPs) stimulate GTP hydrolysis and guanine nucleotide
exchange factors (GEFs) catalyze the GDP-GTP exchange. Rab proteins interact with effector proteins
only in the active state, thereby regulating vesicular trafficking in eukaryotic cells. For this purpose, the
activity and the intracellular localization of Rab proteins need to be tightly regulated. In order to ensure
their own survival, some intracellular pathogens have developed intriguing strategies for manipulation
of intracellular vesicular transport processes and in particular of the Rab proteins involved. A prominent
example of an intracellular pathogen that manipulates Rab proteins for its own benefit is Legionella
pneumophila. In particular, the Legionella protein DrrA (defect in Rab recruitment A) was identified in
the recent past as a protein that manipulates the intracellular localization and activity of Rab1. At the
beginning of this work, structural studies on this protein showed the presence of an additional,
previously uncharacterized domain possessing adenylyltransferase activity towards Rab1. The
characterization of this enzymatic activity was the central subject of this work.
Within this work, the x-ray crystal structure of adenylylated Rab1 was solved. This structure showed that
Rab1 was specifically modified on a tyrosine residue in the functionally important switch II region.
Further studies of the effects of this modification showed that the interaction of Rab1-AMP with GAPs
and the human effector Mical-3 are drastically inhibited, whereas the interaction with the GEF domain
of the Legionella protein DrrA and the Legionella effector protein LidA are not significantly inhibited.
Characterisation of the enzyme kinetics of DrrA and the recently identified deadenylylating enzyme SidD
showed that Rab1:GTP is the preferred substrate of adenylylation by DrrA while SidD possesses a
significantly lower substrate specificity towards the active or inactive conformation of Rab1. This work
includes the first description and characterization of adenylylation as a posttranslational modification of
Rab proteins. In the context of the current literature, the results of this work allowed the proposal of a
model in which adenylylation temporarily inhibits deactivation of Rab1 by GAPs and thus the extraction
of Rab1 from the Legionella containing vacuolar (LCV) membrane by GDI and Rab1 is entrapped at the
LCV membrane. At a later stage of infection, deadenylylation by SidD allows for deactivation and
extraction by GDI. Less
The APPL and APPL proteins APPL adaptor protein phosphotyrosine interaction pleckstrin homology PH domain and leucine zipper-containing protein are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus They play important roles in signal transduction through their ability to act as Rab effectors Rabs are a family of Ras GTPases involved in membrane trafficking Both APPL and APPL comprise an N-terminal membrane-curving BAR Bin-amphiphysin-Rvs domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain The structure and interactions of APPL are well characterized ... More
The APPL1 and APPL2 proteins (APPL (adaptor protein, phosphotyrosine interaction, pleckstrin homology (PH) domain, and leucine zipper-containing protein)) are localized to their own endosomal subcompartment and interact with a wide range of proteins and small molecules at the cell surface and in the nucleus. They play important roles in signal transduction through their ability to act as Rab effectors. (Rabs are a family of Ras GTPases involved in membrane trafficking.) Both APPL1 and APPL2 comprise an N-terminal membrane-curving BAR (Bin-amphiphysin-Rvs) domain linked to a PH domain and a C-terminal phosphotyrosine-binding domain. The structure and interactions of APPL1 are well characterized, but little is known about APPL2. Here, we report the crystal structure and low resolution solution structure of the BARPH domains of APPL2. We identify a previously undetected hinge site for rotation between the two domains and speculate that this motion may regulate APPL2 functions. We also identified Rab binding partners of APPL2 and show that these differ from those of APPL1, suggesting that APPL-Rab interaction partners have co-evolved over time. Isothermal titration calorimetry data reveal the interaction between APPL2 and Rab31 has a Kd of 140 nm. Together with other biophysical data, we conclude the stoichiometry of the complex is 2:2. Less
Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV- neutralization Here we report an HIV- gp membrane-proximal external region MPER -specific antibody named E which neutralizes of tested viruses An analysis of sera from healthy HIV- -infected donors demonstrated that contained MPER-specific antibodies and contained E -like specificities In contrast to other neutralizing MPER antibodies E did not bind phospholipids was not autoreactive and bound cell-surface envelope The structure of E in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp -hydrophobic residues and a critical Arg Lys ... More
Characterization of human monoclonal antibodies is providing considerable insight into mechanisms of broad HIV-1 neutralization. Here we report an HIV-1 gp41 membrane-proximal external region (MPER)-specific antibody, named 10E8, which neutralizes ~98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 27% contained MPER-specific antibodies and 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site-of-vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues and a critical Arg/Lys just prior to the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. The highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies, suggesting that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 Env. Less
In the recent past macromolecular crystallography has gone through substantial methodological and technological development The purpose of this review is to provide a general overview of structural biology and its impact on enzyme structure function analysis and illustrate how it is modifying the focus of research relevant to alkaloid biosynthesis
PII proteins are central signal processing units for the regulation of nitrogen metabolism in bacteria archaea and plants They act in response to cellular energy carbon and nitrogen availability The central metabolites ATP ADP and -oxoglutarate which indicate cellular energy and carbon nitrogen abundance bind in a highly organized manner to PII and induce effector-molecule-dependent conformational states of the T-loop Depending on these states PII proteins bind and modulate the activity of various regulatory targets A mutant variant of the Synechococcus elongatus PII protein PII-I N has been identified to have impaired - oxoglutarate binding Here the PII-I N variant ... More
PII proteins are central signal processing units for the regulation of nitrogen metabolism in bacteria, archaea and plants. They act in response to cellular energy, carbon and nitrogen availability. The central metabolites ATP, ADP and 2-oxoglutarate, which indicate cellular energy and carbon/nitrogen abundance, bind in a highly organized manner to PII and induce effector-molecule-dependent conformational states of the T-loop. Depending on these states, PII proteins bind and modulate the activity of various regulatory targets. A mutant variant of the Synechococcus elongatus PII protein (PII-I86N) has been identified to have impaired 2-�oxoglutarate binding. Here, the PII-I86N variant was cocrystallized in the presence of ATP, magnesium and citrate and its structure was solved at a resolution of 1.05 �. The PII-I86N variant bound citrate in place of 2-oxoglutarate. Citrate binding is mediated primarily by interactions with the ATP-coordinated magnesium ion and the backbone atoms of the T-�loop. Citrate binding rearranges the conformation of the T-�loop and, consistent with this, citrate suppresses the binding of PII-I86N to an NAG kinase variant, which is similar to the suppression of PII-NAG kinase complex formation by 2-OG. Based on the structures of 2-OG and citrate, homocitrate was suggested as a third ligand and an efficient response towards this molecule with different functional properties was observed. Together, these data provide a first glimpse of a genetically engineered PII variant that senses a new effector molecule. Less
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli purified and crystallized Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen The crystals belonged to the primitive tetragonal space group P with unit-cell parameters a b c Complete data sets were collected from native and selenomethionine-substituted protein crystals at K to and resolution respectively
As with many other viruses the initial cell attachment of rotaviruses which are the major causative agent of infantile gastroenteritis is mediated by interactions with specific cellular glycans The distally located VP domain of the rotavirus spike protein VP ref mediates such interactions The existing paradigm is that sialidase-sensitive animal rotavirus strains bind to glycans with terminal sialic acid Sia whereas sialidase-insensitive human rotavirus strains bind to glycans with internal Sia such as GM ref Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies it is not yet known how VP of human rotaviruses ... More
As with many other viruses, the initial cell attachment of rotaviruses, which are the major causative agent of infantile gastroenteritis, is mediated by interactions with specific cellular glycans1,2,3,4. The distally located VP8* domain of the rotavirus spike protein VP4 (ref. 5) mediates such interactions. The existing paradigm is that ‘sialidase-sensitive’ animal rotavirus strains bind to glycans with terminal sialic acid (Sia), whereas ‘sialidase-insensitive’ human rotavirus strains bind to glycans with internal Sia such as GM1 (ref. 3). Although the involvement of Sia in the animal strains is firmly supported by crystallographic studies1,3,6,7, it is not yet known how VP8* of human rotaviruses interacts with Sia and whether their cell attachment necessarily involves sialoglycans. Here we show that VP8* of a human rotavirus strain specifically recognizes A-type histo-blood group antigen (HBGA) using a glycan array screen comprised of 511 glycans, and that virus infectivity in HT-29 cells is abrogated by anti-A-type antibodies as well as significantly enhanced in Chinese hamster ovary cells genetically modified to express the A-type HBGA, providing a novel paradigm for initial cell attachment of human rotavirus. HBGAs are genetically determined glycoconjugates present in mucosal secretions, epithelia and on red blood cells8, and are recognized as susceptibility and cell attachment factors for gastric pathogens like Helicobacter pylori9 and noroviruses10. Our crystallographic studies show that the A-type HBGA binds to the human rotavirus VP8* at the same location as the Sia in the VP8* of animal rotavirus, and suggest how subtle changes within the same structural framework allow for such receptor switching. These results raise the possibility that host susceptibility to specific human rotavirus strains and pathogenesis are influenced by genetically controlled expression of different HBGAs among the world’s population. Less
Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input To investigate the conformational changes that relay the regulatory signal we have studied the HAMP domain a ubiquitous intracellular module connecting input to output domains HAMP forms a parallel dimeric four-helical coiled coil and rational substitutions in our model domain Af HAMP induce a transition in its interhelical packing characterized by axial rotation of all four helices the gearbox signaling model We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af HAMP variants to the DHp domain of EnvZ a ... More
Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity. Less
Cytoskeletal intermediate filaments IFs assemble from the elementary dimers based on a segmented -helical coiled-coil CC structure Crystallographic studies of IF protein fragments remain the main route to access their atomic structure To enable crystallization such fragments must be sufficiently short As a consequence they often fail to assemble into the correct CC dimers In particular human vimentin fragment D corresponding to the first half of coil residues stays monomeric in solution We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus The crystal structure ... More
Cytoskeletal intermediate filaments (IFs) assemble from the elementary dimers based on a segmented α-helical coiled-coil (CC) structure. Crystallographic studies of IF protein fragments remain the main route to access their atomic structure. To enable crystallization, such fragments must be sufficiently short. As a consequence, they often fail to assemble into the correct CC dimers. In particular, human vimentin fragment D3 corresponding to the first half of coil2 (residues 261–335) stays monomeric in solution. We have induced its dimerization via introducing a disulfide link between two cysteines engineered in the hydrophobic core of the CC close to its N-terminus. The 2.3 Å crystal structure of the D3st (stabilized) fragment reveals a mostly parallel α-helical bundle structure in its N-terminal half which smoothly continues into a left-handed CC towards the C-terminus. This provides a direct evidence for a continuously α-helical structure of the coil2 segment and disproves the previously suggested existence of linker L2 separating it into two left-handed CCs. The general principles of CC dimer stabilization by disulfide introduction are also discussed. Less
A broad working definition of structural proteomics SP is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules Recently the process for protein structure determination has become highly automated and SP platforms have been established around the globe utilizing X-ray crystallography as a tool Although protein structures often provide clues about the biological function of a target once the three-dimensional structures have been determined bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles This article reviews the current status of SP methods for the structure determination pipeline ... More
A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation. Less
The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein There is no good reason however why this -carbon cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness intrinsic curvature lateral pressure profile lipid and protein makeup and compositional asymmetry Thus it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile For this monoacylglycerols with differing acyl chain characteristics such ... More
The default lipid for the bulk of the crystallogenesis studies performed to date using the cubic mesophase method is monoolein. There is no good reason however, why this 18-carbon, cis-monounsaturated monoacylglycerol should be the preferred lipid for all target membrane proteins. The latter come from an array of biomembrane types with varying properties that include hydrophobic thickness, intrinsic curvature, lateral pressure profile, lipid and protein makeup, and compositional asymmetry. Thus, it seems reasonable that screening for crystallizability based on the identity of the lipid creating the hosting mesophase would be worthwhile. For this, monoacylglycerols with differing acyl chain characteristics, such as length and olefinic bond position, must be available. A lipid synthesis and purification program is in place in the author's laboratory to serve this need. In the current study with the outer membrane sugar transporter, OprB, we demonstrate the utility of host lipid screening as a means for generating diffraction-quality crystals. Host lipid screening is likely to prove a generally useful strategy for mesophase-based crystallization of membrane proteins. Less
The flax rust effector AvrM is a secreted protein of unknown fold that is recognized by the M resistance protein in flax In order to investigate the structural basis of the AvrM M interaction and possible virulence-associated functions of AvrM the C-terminal domains of two different AvrM variants AvrM-A and avrM were crystallized Crystals of native AvrM-A were obtained using pentaerythritol ethoxylate EO OH as a precipitant and diffracted X-rays to resolution Selenomethionine-derivative crystals of similar quality were obtained using PEG as a precipitant Both the native and selenomethionine-labelled AvrM-A crystals had symmetry of space group C with eight molecules ... More
The flax rust effector AvrM is a secreted protein of unknown fold that is recognized by the M resistance protein in flax. In order to investigate the structural basis of the AvrM�M interaction and possible virulence-associated functions of AvrM, the C-terminal domains of two different AvrM variants (AvrM-A and avrM) were crystallized. Crystals of native AvrM-A were obtained using pentaerythritol ethoxylate (15/4 EO/OH) as a precipitant and diffracted X-rays to 2.9 � resolution. Selenomethionine-derivative crystals of similar quality were obtained using PEG 1500 as a precipitant. Both the native and selenomethionine-labelled AvrM-A crystals had symmetry of space group C2221 with eight molecules in the asymmetric unit. Crystals of avrM had symmetry of space group P212121 and diffracted X-rays to 2.7 � resolution. Initial AvrM-A phases were calculated using the single-wavelength anomalous dispersion (SAD) method and a partial model was built. Phases for avrM were obtained by molecular replacement using the partial AvrM-A model. Less
AAA proteins are ATPases associated with diverse cellular activities coupling ATP-hydrolysis to remodelling disaggregation and unfolding of a variety of substrates The central ATPase domain functions as a molecular switch which receives input from N-terminal substrate recognition domains and which transfers the output to downstream effectors AAA proteases recognize misfolded proteins with their N-domain unfold and thread them through the pore of the hexameric ring and feed them to proteases either residing on the same polypeptide chain or being contacted via C-terminal interaction motifs
Susceptibility to norovirus NoV a major pathogen of epidemic gastroenteritis is associated with histo-blood group antigens HBGAs which are also cell attachment factors for this virus GII NoV strains are predominantly associated with worldwide NoV epidemics with a periodic emergence of new variants The sequence variations in the surface-exposed P domain of the capsid protein resulting in differential HBGA binding patterns and antigenicity are suggested to drive GII epochal evolution To understand how temporal sequence variations affect the P domain structure and contribute to epochal evolution we determined the P domain structure of a variant with ABH and secretor Lewis ... More
Susceptibility to norovirus (NoV), a major pathogen of epidemic gastroenteritis, is associated with histo-blood group antigens (HBGAs), which are also cell attachment factors for this virus. GII.4 NoV strains are predominantly associated with worldwide NoV epidemics with a periodic emergence of new variants. The sequence variations in the surface-exposed P domain of the capsid protein resulting in differential HBGA binding patterns and antigenicity are suggested to drive GII.4 epochal evolution. To understand how temporal sequence variations affect the P domain structure and contribute to epochal evolution, we determined the P domain structure of a 2004 variant with ABH and secretor Lewis HBGAs and compared it with the previously determined structure of a 1996 variant. We show that temporal sequence variations do not affect the binding of monofucosyl ABH HBGAs but that they can modulate the binding strength of difucosyl Lewis HBGAs and thus could contribute to epochal evolution by the potentiated targeting of new variants to Lewis-positive, secretor-positive individuals. The temporal variations also result in significant differences in the electrostatic landscapes, likely reflecting antigenic variations. The proximity of some of these changes to the HBGA binding sites suggests the possibility of a coordinated interplay between antigenicity and HBGA binding in epochal evolution. From the observation that the regions involved in the formation of the HBGA binding sites can be conformationally flexible, we suggest a plausible mechanism for how norovirus disassociates from salivary mucin-linked HBGA before reassociating with HBGAs linked to intestinal epithelial cells during its passage through the gastrointestinal tract. Less
Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown Here we present the crystal structure of human dynamin in the nucleotide-free state with a four-domain architecture comprising the GTPase domain the bundle signalling element the stalk and the pleckstrin homology domain Dynamin oligomerized in the crystals via the stalks which assemble in a criss-cross fashion The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of ... More
Dynamin is a mechanochemical GTPase that oligomerizes around the neck of clathrin-coated pits and catalyses vesicle scission in a GTP-hydrolysis-dependent manner. The molecular details of oligomerization and the mechanism of the mechanochemical coupling are currently unknown. Here we present the crystal structure of human dynamin 1 in the nucleotide-free state with a four-domain architecture comprising the GTPase domain, the bundle signalling element, the stalk and the pleckstrin homology domain. Dynamin 1 oligomerized in the crystals via the stalks, which assemble in a criss-cross fashion. The stalks further interact via conserved surfaces with the pleckstrin homology domain and the bundle signalling element of the neighbouring dynamin molecule. This intricate domain interaction rationalizes a number of disease-related mutations in dynamin 2 and suggests a structural model for the mechanochemical coupling that reconciles previous models of dynamin function. Less
The lipidic cubic phase LCP has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins While monoolein has been the predominant lipid of choice there is a growing need for the characterization and use of other LCP host lipids allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion stability and crystallogenesis of membrane proteins Here we describe the development of a high-throughput HT pipeline to employ small angle X-ray scattering SAXS the most direct technique to identify lipid mesophases and measure their structural parameters to ... More
The lipidic cubic phase (LCP) has repeatedly proven to serve as a successful membrane-mimetic matrix for a variety of difficult-to-crystallize membrane proteins. While monoolein has been the predominant lipid of choice, there is a growing need for the characterization and use of other LCP host lipids, allowing exploration of a range of structural parameters such as bilayer thickness and curvature for optimal insertion, stability and crystallogenesis of membrane proteins. Here, we describe the development of a high-throughput (HT) pipeline to employ small angle X-ray scattering (SAXS) � the most direct technique to identify lipid mesophases and measure their structural parameters � to interrogate rapidly a large number of lipid samples under a variety of conditions, similar to those encountered during crystallization. Leveraging the identical setup format for LCP crystallization trials, this method allows the quickly assessment of lipid matrices for their utility in membrane protein crystallization, and could inform the tailoring of lipid and precipitant conditions to overcome specific crystallization challenges. As proof of concept, we present HT LCP-SAXS analysis of lipid samples made of monoolein with and without cholesterol, and of monovaccenin, equilibrated with solutions used for crystallization trials and LCP fluorescence recovery after photobleaching (FRAP) experiments. Less
A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and in a high-throughput environment the development of robotic systems for storing and imaging crystallization trials Most of these trials are carried out in -well or higher density plates and managing them is a significant information management challenge We describe xtalPiMS a web-based application for the management and monitoring of crystallization trials xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System PiMS and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization ... More
A major advance in protein structure determination has been the advent of nanolitre-scale crystallization and (in a high-throughput environment) the development of robotic systems for storing and imaging crystallization trials. Most of these trials are carried out in 96-well (or higher density) plates and managing them is a significant information management challenge. We describe xtalPiMS, a web-based application for the management and monitoring of crystallization trials. xtalPiMS has a user-interface layer based on the standards of the Protein Information Management System (PiMS) and a database layer which links the crystallization trial images to the meta-data associated with a particular crystallization trial. The user interface has been optimized for the efficient monitoring of high-throughput environments with three different automated imagers and work to support a fourth imager is in progress, but it can even be of use without robotics. The database can either be a PiMS database or a legacy database for which a suitable mapping layer has been developed. Less
The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation This process is catalyzed by members of the heme-copper oxidase HCO superfamily Despite the availability of crystal structures for all types of HCO the mode of action for this enzyme is not understood at the atomic level namely how vectorial H and e- transport are coupled Toward addressing this problem we report wild type and A F mutant structures of the ba -type cytochrome c oxidase from Thermus thermophilus at resolution The enzyme has been crystallized from the lipidic cubic phase which ... More
The fundamental chemistry underpinning aerobic life on Earth involves reduction of dioxygen to water with concomitant proton translocation. This process is catalyzed by members of the heme-copper oxidase (HCO) superfamily. Despite the availability of crystal structures for all types of HCO, the mode of action for this enzyme is not understood at the atomic level, namely how vectorial H+ and e- transport are coupled. Toward addressing this problem, we report wild type and A120F mutant structures of the ba3-type cytochrome c oxidase from Thermus thermophilus at 1.8 � resolution. The enzyme has been crystallized from the lipidic cubic phase, which mimics the biological membrane environment. The structures reveal 20 ordered lipid molecules that occupy binding sites on the protein surface or mediate crystal packing interfaces. The interior of the protein encloses 53 water molecules, including 3 trapped in the designated K-path of proton transfer and 8 in a cluster seen also in A-type enzymes that likely functions in egress of product water and proton translocation. The hydrophobic O2-uptake channel, connecting the active site to the lipid bilayer, contains a single water molecule nearest the CuB atom but otherwise exhibits no residual electron density. The active site contains strong electron density for a pair of bonded atoms bridging the heme Fea3 and CuB atoms that is best modeled as peroxide. The structure of ba3-oxidase reveals new information about the positioning of the enzyme within the membrane and the nature of its interactions with lipid molecules. The atomic resolution details provide insight into the mechanisms of electron transfer, oxygen diffusion into the active site, reduction of oxygen to water, and pumping of protons across the membrane. The development of a robust system for production of ba3-oxidase crystals diffracting to high resolution, together with an established expression system for generating mutants, opens the door for systematic structure-function studies. Less
The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations Histamine-H receptor H R antagonists are very effective drugs alleviating the symptoms of allergic reactions Here we show the crystal structure of H R complex with doxepin a first-generation H R-antagonist Doxepin sits deep in the ligand binding pocket and directly interacts with the highly conserved Trp a key residue in GPCR activation This well-conserved pocket with mostly hydrophobic nature contributes to low selectivity of the first-generation compounds The pocket is associated with an anion-binding region occupied by a phosphate ion Docking ... More
The biogenic amine histamine is an important pharmacological mediator involved in pathophysiological processes such as allergies and inflammations. Histamine-H1 receptor (H1R) antagonists are very effective drugs alleviating the symptoms of allergic reactions. Here we show the crystal structure of H1R complex with doxepin, a first-generation H1R-antagonist. Doxepin sits deep in the ligand binding pocket and directly interacts with the highly conserved Trp4286.48, a key residue in GPCR activation. This well-conserved pocket with mostly hydrophobic nature contributes to low selectivity of the first-generation compounds. The pocket is associated with an anion-binding region occupied by a phosphate ion. Docking of various second-generation H1R-antagonists reveals that the unique carboxyl-group present in this class of compounds interacts with Lys1915.39 and/or Lys179ECL2, both of which form part of the anion-binding region. This region is not conserved in other aminergic receptors defining how minor differences in receptor lead to pronounced selectivity differences with small molecules. Less
Many bacteria kill related bacteria by secretion of bacteriocins In Escherichia coli the colicin M protein kills E coli after uptake into the periplasm Self-protection from destruction is provided by the co-expressed immunity protein The colicin M immunity protein Cmi was cloned overexpressed and purified to homogeneity The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy Crystallization trials yielded crystals one of which diffracted to a resolution of in the orthorhombic space group C The crystal packing with unit-cell parameters a b c indicated the presence of one monomer in the asymmetric unit with a ... More
Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 � in the orthorhombic space group C2221. The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 �, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%. Less
HAMP domains mediate signal transduction in over enzyme-coupled receptors represented in all kingdoms of life The HAMP domain of the putative archaeal receptor Af has a parallel dimeric four-helical coiled coil structure but with unusual core packing related to canonical packing by concerted axial rotation of the helices This has led to the gearbox model for signal transduction whereby the alternate packing modes correspond to signaling states Here we present structures of a series of Af HAMP variants We show that substitution of a conserved small side chain within the domain core A for larger residues induces a gradual transition ... More
HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems. Less
Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids It functions in part by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations The peptide should be able to traverse the host membrane either as a double stranded intertwined double helix DSDH or as a head-to-head single stranded helix HHSH Current structure models are based on macromolecular X-ray crystallography MX and nuclear magnetic resonance NMR However the HHSH form has only been observed by NMR The shape and size of the different gramicidin conformations differ We speculated therefore that reconstituting it into a ... More
Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation. Less
The Toll interleukin- receptor TIR domain is a protein protein interaction domain that is found in both animal and plant immune receptors In animal Toll-like receptor signalling both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response The TIR domains in cytoplasmic nucleotide-binding leucine-rich repeat NB-LRR plant disease-resistance proteins are not as well characterized but recent studies have suggested a role in defence signalling In this study the crystallization X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein ... More
The Toll/interleukin-1 receptor (TIR) domain is a protein�protein interaction domain that is found in both animal and plant immune receptors. In animal Toll-like receptor signalling, both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response. The TIR domains in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant disease-resistance proteins are not as well characterized, but recent studies have suggested a role in defence signalling. In this study, the crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6 (L6TIR) are reported. Plate-like crystals of L6TIR were obtained using PEG 200 as a precipitant and diffracted X-rays to 2.3 � resolution. Pseudo-translation complicated the initial assignment of the crystal symmetry, which was ultimately found to correspond to space group P21212 with two molecules per asymmetric unit. The structure of L6TIR was solved by molecular replacement using the structure of the TIR-domain-containing protein AT1G72930 from Arabidopsis as a template. Less
G protein-coupled receptors GPCRs constitute a highly diverse and ubiquitous family of integral membrane proteins transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase LCP The success of ... More
G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus, generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success. Less
Colicin M Cma is specifically imported into the periplasm of Escherichia coli and kills the cells Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase chaperone FkpA To identify the Cma prolyl bonds targeted by FkpA we replaced the proline residues individually with alanine Seven mutant proteins were fully active Cma P A Cma P A and Cma P A displayed and Cma P A displayed of the wild-type activity Cma P A Cma P A and Cma P A but not Cma P A killed cells after entering the periplasm via osmotic shock indicating that the former mutants were ... More
Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA. Less
In Escherichia coli the -barrel assembly machinery or BAM complex mediates the recognition insertion and assembly of outer membrane proteins The complex consists of the integral membrane protein BamA an Omp -family member and the lipoproteins BamB BamC BamD and BamE The purification and crystallization of BamC BamD and BamE each lacking the N- terminal membrane anchor is described While the smallest protein BamE yielded crystals under conventional conditions BamD only crystallized after stabilization with urea Full-length BamC did not crystallize but was cleaved by subtilisin into two domains which were subsequently crystallized independently High-resolution data were acquired from all ... More
In Escherichia coli, the �-barrel assembly machinery (or BAM complex) mediates the recognition, insertion and assembly of outer membrane proteins. The complex consists of the integral membrane protein BamA (an Omp85-family member) and the lipoproteins BamB, BamC, BamD and BamE. The purification and crystallization of BamC, BamD and BamE, each lacking the N-�terminal membrane anchor, is described. While the smallest protein BamE yielded crystals under conventional conditions, BamD only crystallized after stabilization with urea. Full-length BamC did not crystallize, but was cleaved by subtilisin into two domains which were subsequently crystallized independently. High-resolution data were acquired from all proteins. Less