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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
Structural studies of human G protein-coupled receptors GPCRs have recently been accelerated through the use of the T lysozyme fusion partner that was inserted into the third intracellular loop Using chimeras of the human -adrenergic and human A A adenosine receptors we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs In particular the use of the thermostabilized apocytochrome b RIL as a fusion partner displays certain advantages over the previously utilized T lysozyme resulting in a significant improvement in stability and structure in GPCR-fusion constructs
Members of the opioid receptor family of G-protein-coupled receptors GPCRs are found throughout the peripheral and central nervous system where they have key roles in nociception and analgesia Unlike the classical opioid receptors and -OR -OR and -OR which were delineated by pharmacological criteria in the s and s the nociceptin orphanin FQ N OFQ peptide receptor NOP also known as ORL- was discovered relatively recently by molecular cloning and characterization of an orphan GPCR Although it shares high sequence similarity with classical opioid GPCR subtypes NOP has a markedly distinct pharmacology featuring activation by the endogenous peptide N OFQ ... More
Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the ‘classical’ opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR1. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands2,3. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP–compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands. Less
Second-order nonlinear optical imaging of chiral crystals SONICC is an emerging technique for crystal imaging and characterization We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications Given that they provide near-complete suppression of any background SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis Because SONICC creates high-resolution images nucleation and growth kinetics can also be observed SONICC can detect metastable homochiral crystalline forms of amino acids crystallizing from racemic solutions which confirms ... More
Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald�s rule of stages for crystal growth. SONICC�s selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation. Less
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps This has enabled the crystallization of large numbers of proteins including integral membrane proteins This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality The latter underscores the importance of the ... More
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps. This has enabled the crystallization of large numbers of proteins, including integral membrane proteins. This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success. Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality. The latter underscores the importance of the development of powerful biophysical characterization techniques. Less
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
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes including the regulation of pain respiratory drive mood and in the case of -opioid receptor -OR dysphoria and psychotomimesis Here we report the crystal structure of the human -OR in complex with the selective antagonist JDTic arranged in parallel dimers at resolution The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human -OR Modelling of other important -OR-selective ligands including the morphinan-derived antagonists norbinaltorphimine and -guanidinonaltrindole and the diterpene agonist salvinorin A analogue ... More
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. 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 crystal structure of LsrB from Yersinia pestis complexed with autoinducer- AI- space group P unit-cell parameters a b c has been solved by molecular replacement using the structure of LsrB from Salmonella typhimurium PDB entry tjy and refined to R R free at resolution The electron density for bound AI- and the stereochemistry of the AI- -binding site are consistent with bound AI- adopting the R S - -methyl- -tetrahydroxytetrahydrofuran conformation just as has been observed in the crystal structures of the Salmonella typhimurium and Sinorhizobium meliloti LsrB AI- complexes
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
Second order nonlinear optical imaging of chiral crystals SONICC is a promising new method for the sensitive and selective detection of protein crystals Relevant general principles of second harmonic generation which underpins SONICC are reviewed Instrumentation and methods for SONICC measurements are described and critically assessed in terms of performance trade-offs Potential origins of false-positives and false-negatives are also discussed
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
Lipidic cubic phase LCP is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment LCP technologies however have not been fully embraced by the membrane protein structural biology community primarily because of the difficulties associated with handling viscous materials Recent developments of pre-crystallization assays and improvements in crystal imaging successes in obtaining high resolution structures of G protein-coupled receptors GPCRs and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein ... More
Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions. 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
The primary bottleneck in synthetic biology research today is the construction of physical DNAs a process that is often expensive time-consuming and riddled with cloning difficulties associated with the uniqueness of each DNA sequence We have developed a series of biological and computational tools that lower existing barriers to automation and scaling to enable affordable fast and accurate construction of large DNA sets Here we provide detailed protocols for high-throughput automated assembly of BglBrick standard biological parts using iterative ab reactions We have implemented these protocols on a minimal hardware platform consisting of a Biomek liquid handling robot a benchtop ... More
The primary bottleneck in synthetic biology research today is the construction of physical DNAs, a process that is often expensive, time-consuming, and riddled with cloning difficulties associated with the uniqueness of each DNA sequence. We have developed a series of biological and computational tools that lower existing barriers to automation and scaling to enable affordable, fast, and accurate construction of large DNA sets. Here we provide detailed protocols for high-throughput, automated assembly of BglBrick standard biological parts using iterative 2ab reactions. We have implemented these protocols on a minimal hardware platform consisting of a Biomek 3000 liquid handling robot, a benchtop centrifuge and a plate thermocycler, with additional support from a software tool called AssemblyManager. This methodology enables parallel assembly of several hundred large error-free DNAs with a 96+% success rate. 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
P II proteins control key processes of nitrogen metabolism in bacteria archaea and plants in response to the central metabolites ATP ADP and -oxoglutarate -OG signaling cellular energy and carbon and nitrogen abundance This metabolic information is integrated by P II and transmitted to regulatory targets key enzymes transporters and transcription factors modulating their activity In oxygenic phototrophs the controlling enzyme of arginine synthesis N-acetyl-glutamate kinase NAGK is a major P II target whose activity responds to -OG via P II Here we show structures of the Synechococcus elongatus P II protein in complex with ATP Mg and -OG which ... More
P II proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P II and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P II target, whose activity responds to 2-OG via P II . Here we show structures of the Synechococcus elongatus P II protein in complex with ATP, Mg??, and 2-OG, which clarify how 2-OG affects P II -NAGK interaction. P II trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P II trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg?? ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P II target. Structures of P II trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P II senses effectors in a wide range of concentrations. Less
Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components however the number of these targets is limited In the search for new targets and lead compounds C neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase IMPDH in contrast a rare subtype of the related C gattii is naturally resistant Here the expression purification crystallization and preliminary crystallographic analysis of IMPDH ... More
Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals. The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components; however, the number of these targets is limited. In the search for new targets and lead compounds, C. neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase (IMPDH); in contrast, a rare subtype of the related C. gattii is naturally resistant. Here, the expression, purification, crystallization and preliminary crystallographic analysis of IMPDH complexed with IMP and NAD+ is reported for both of these Cryptococcus species. The crystals of IMPDH from both sources had the symmetry of the tetragonal space group I422 and diffracted to a resolution of 2.5 Å for C. neoformans and 2.6 Å for C. gattii. Less
PII signal transduction proteins are highly conserved in bacteria archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon nitrogen and energy status of the cell In the cyanobacterium Synechococcus elongatus PCC PII binds ATP and -oxoglutarate -OG in a synergistic manner with the ATP binding sites also accepting ADP Depending on its effector molecule binding status PII from this cyanobacterium and other oxygenic phototrophs complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway N-acetyl-l-glutamate kinase NAGK to control arginine biosynthesis To gain deeper insights into the process of PII ... More
PII signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, PII binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, PII (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of PII binding to NAGK, we searched for PII variants with altered binding characteristics and found PII variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type PII protein. Analysis of interactions between these PII variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the PII I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the PII I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of PII proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus PII after having latched onto NAGK. Moreover, both PII I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of PII–NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of PII initiates the bending of the extended T-loop of PII, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. Less
Pathogens require protein-folding enzymes to produce functional virulence determinants These foldases include the Dsb family of proteins which catalyze oxidative folding in bacteria Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K- and these mechanisms have been extrapolated to other organisms However recent research indicates that the K- complement of Dsb proteins is not common to all bacteria Importantly many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins Salmonella enterica serovar Typhimurium ... More
Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-�12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 � and belonged to space groups P21, P21212 and C2, respectively. Less
The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence epi-illumination or absorbance transmission are evaluated In agreement with other studies tryptophan residues are found on average to be largely buried in protein structures with of their surface area buried and to be surrounded by partially polar microenvironments with of their surface area covered by polar residues which suggests an inherent degree of fluorescence signal quenching In bacterial genomes up to one-third on average of open reading frames are deficient in tryptophan In ... More
The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with ~84% of their surface area buried) and to be surrounded by partially polar microenvironments (with ~43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (~18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed. Less
Second order nonlinear optical imaging of chiral crystals SONICC is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and or lipids that often form various mesophases Detection of crystals in such media by conventional optical methods e g intrinsic UV fluorescence birefringence bright-field image analysis etc is often complicated by optical scattering and by the small sizes of the crystals that routinely form SONICC is shown to be well-suited for this application by nature of its compatibility ... More
Second order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 micron) with a minimal background due to the surrounding lipid mesophase (~1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed. Less
The use of design of experiments DOE in assay development AD has the potential to speed up assay optimisation ie reduce assay development bottlenecks and to facilitate a more thorough evaluation of assay variables Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays Although standalone DOE software packages are available these were not written specifically for biological applications and they vary in their suitability for AD DOE needs to be simpler to implement to make a major impact on AD A market opportunity exists for a turnkey solution that directly links ... More
The use of design of experiments (DOE) in assay development (AD) has the potential to speed up assay optimisation (ie reduce assay development bottlenecks) and to facilitate a more thorough evaluation of assay variables. Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays. Although standalone DOE software packages are available, these were not written specifically for biological applications and they vary in their suitability for AD. DOE needs to be simpler to implement to make a major impact on AD. A market opportunity exists for a turnkey solution that directly links statistical design with automated liquid handler programming and also feeds the assay readout directly into the statistical analysis, to suggest and facilitate further iterative retesting. Until new tools or more encompassing solutions emerge, the full impact of DOE on AD is unlikely to be realised. Less
Background Protein crystallization screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals Generally condition screening is performed in -well plates While previous studies have examined the effects of protein construct protein purity or crystallisation condition ingredients on protein crystallisation few have examined the effect of the crystallisation plate Methodology Principal Findings We performed a statistically rigorous examination of protein crystallisation and evaluated interactions between crystallisation success and plate row column different plates of same make different plate makes and different ... More
Background Protein crystallization screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. Methodology/Principal Findings We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. Conclusions/Significance Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallise, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make. Less
T-cell receptors TCRs are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein Here we report our systematic approach for generating soluble -TCRs for X-ray crystallographic studies By using small-scale expression screens novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database Our success in crystallizing both isolated TCRs and Major histocompatibility complex MHC TCR complexes has provided us with sufficient data ... More
T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, αβ-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes. Less
RNA silencing is a conserved regulatory mechanism in fungi plants and animals that regulates gene expression and defence against viruses and transgenes Small silencing RNAs of nucleotides and their associated effector proteins the Argonaute family proteins are the central components in RNA silencing A subset of small RNAs such as microRNAs and small interfering RNAs siRNAs in plants Piwi-interacting RNAs in animals and siRNAs in Drosophila requires an additional crucial step for their maturation that is '-O-methylation on the ' terminal nucleotide A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase HUA ENHANCER HEN and its homologues are responsible for this specific modification Here ... More
RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ~20�30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide3�6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3�5,7,8. Here we report the 3.1 � crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2'-O-methylation mechanism. Less
Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals Advent of the sub- m minibeam at the APS GM CA CAT has enabled the collection of high quality diffraction data from such microcrystals Herein we describe the challenges and solutions related to growing manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner The resulting diffraction patterns have ... More
Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 �m minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets. Less
A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described This has variously been referred to as the lipid cubic phase or in meso method The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins proteins that are monomeric homo- and hetero-multimeric chromophore-containing and chromophore-free and a-helical and -barrel proteins Its most recent successes are the human engineered -adrenergic and adenosine A A G protein-coupled receptors Protocols are provided for preparing and characterizing the lipidic mesophase for reconstituting the protein ... More
A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and a-helical and �-barrel proteins. Its most recent successes are the human engineered �2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. Less
VERNALIZATION VRN is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment Stable suppression of FLC accelerates flowering a physiological process known as vernalization VRN is a -residue DNA-binding protein that contains two plant-specific B domains B a and B b a putative nuclear localization sequence NLS and two putative PEST domains VRN includes the second B domain and a region upstream that is highly conserved in the VRN orthologues of other dicotyledonous plants VRN was crystallized by the hanging-drop method in M sodium acetate pH containing M NaCl ... More
VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1208�341 includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1208�341 was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1208�341 crystal diffracted to 2.1 � and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 �, a = 90.0, � = 115.4, ? = 90.0�. Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 �3 Da-1 and a solvent content of 40.1% were calculated. Less
The stimulatory RNA of the Visna-Maedi virus VMV - ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix reminiscent of the HIV- frameshift-stimulatory RNA or as an RNA pseudoknot The pseudoknot is unusual in that it would include a nucleotide loop termed here an interstem element ISE between the two stems In almost all frameshift-promoting pseudoknots ISEs are absent or comprise a single adenosine residue Using a combination of RNA structure probing site directed mutagenesis NMR and phylogenetic sequence comparisons we show here that the VMV stimulatory RNA is indeed a pseudoknot ... More
The stimulatory RNA of the Visna-Maedi virus (VMV) -1 ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix, reminiscent of the HIV-1 frameshift-stimulatory RNA, or as an RNA pseudoknot. The pseudoknot is unusual in that it would include a 7 nucleotide loop (termed here an interstem element [ISE]) between the two stems. In almost all frameshift-promoting pseudoknots, ISEs are absent or comprise a single adenosine residue. Using a combination of RNA structure probing, site directed mutagenesis, NMR, and phylogenetic sequence comparisons, we show here that the VMV stimulatory RNA is indeed a pseudoknot, conforming closely to the modeled structure, and that the ISE is essential for frameshifting. Pseudoknot function was predictably sensitive to changes in the length of the ISE, yet altering its sequence to alternate pyrimidine/purine bases was also detrimental to frameshifting, perhaps through modulation of local tertiary interactions. How the ISE is placed in the context of an appropriate helical junction conformation is not known, but its presence impacts on other elements of the pseudoknot, for example, the necessity for a longer than expected loop 1. This may be required to accommodate an increased flexibility of the pseudoknot brought about by the ISE. In support of this, 1H NMR analysis at increasing temperatures revealed that stem 2 of the VMV pseudoknot is more labile than stem 1, perhaps as a consequence of its connection to stem 1 solely via flexible single-stranded loops. Less
In this article we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog We also explain the structure-activity relationship of several recently reported inhibitors The native enzyme crystals were of poor quality they only diffracted X-rays to resolution Therefore we have executed a rational surface mutagenesis strategy that has yielded crystals of this -amino acid multidomain protein diffracting to or better This methodology is discussed and contrasted with the more traditional ... More
In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality—they only diffracted X-rays to 3–5 Å resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 Å or better. This methodology is discussed and contrasted with the more traditional domain truncation approach. Less