TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3 and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 Å and 2.5 Å resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride (PMSF) and paraoxon were determined to 2.4 Å and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which this Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.
Acetyl esterase; Thermotoga maritima; crystal structure; α/β hydrolase; inhibitor; serine rotation
The tad (tight adherence) locus encodes a protein translocation system that produces a novel variant of type IV pili. The pilus assembly protein TadZ (called CpaE in Caulobacter crescentus) is ubiquitous in tad loci, but is absent in other type IV pilus biogenesis systems. The crystal structure of TadZ from E. rectale (ErTadZ), in complex with ATP and Mg2+, was determined to 2.1 Å resolution. ErTadZ contains an atypical ATPase domain with a variant of a deviant Walker-A motif that retains ATP binding capacity while displaying only low intrinsic ATPase activity. The bound ATP plays an important role in dimerization of ErTadZ. The N-terminal atypical receiver domain resembles the canonical receiver domain of response regulators, but has a degenerate, stripped-down “active site”. Homology modeling of the N-terminal atypical receiver domain of CpaE indicates that it has a conserved protein-protein binding surface similar to that of the polar localization module of the social mobility protein FrzS, suggesting a similar function. Our structural results also suggest that TadZ localizes to the pole through the atypical receiver domain during early stage of pili biogenesis, and functions as a hub for recruiting other pili components, thus providing insights into the Tad pilus assembly process.
Type IV pili assembly; TadZ; atypical receiver domain; atypical ATPase; localization factor
MtfA of Escherichia coli (formerly YeeI) was previously identified as a regulator of the phosphoenolpyruvate (PEP)-dependent:glucose phosphotransferase system. MtfA homolog proteins are highly conserved, especially among beta- and gammaproteobacteria. We determined the crystal structures of the full-length MtfA apoenzyme from Klebsiella pneumoniae and its complex with zinc (holoenzyme) at 2.2 and 1.95 Å, respectively. MtfA contains a conserved H149E150XXH153+E212+Y205 metallopeptidase motif. The presence of zinc in the active site induces significant conformational changes in the region around Tyr205 compared to the conformation of the apoenzyme. Additionally, the zinc-bound MtfA structure is in a self-inhibitory conformation where a region that was disordered in the unliganded structure is now observed in the active site and a nonproductive state of the enzyme is formed. MtfA is related to the catalytic domain of the anthrax lethal factor and the Mop protein involved in the virulence of Vibrio cholerae, with conservation in both overall structure and in the residues around the active site. These results clearly provide support for MtfA as a prototypical zinc metallopeptidase (gluzincin clan).
The crystal structures of an unliganded and adenosine 5′-monophosphate (AMP) bound, metal-dependent phosphoesterase (YP_910028.1) from Bifidobacterium adolescentis are reported at 2.4 Å and 1.94 Å, respectively. Functional characterization of this enzyme was guided by computational analysis and then confirmed by experiment. The structure consists of a PHP (Polymerase and Histidinol Phosphatase, Pfam: PF02811) domain with a second domain (residues 105–178) inserted in the middle of the PHP sequence. The insert domain functions in binding AMP, but the precise function and substrate specificity of this domain is unknown. Initial bioinformatics analyses yielded multiple potential functional leads, with most of them suggesting DNA polymerase or DNA replication activity. Phylogenetic analysis indicated a potential DNA polymerase function that was somewhat supported by global structural comparisons identifying the closest structural match to the alpha subunit of DNA polymerase III. However, several other functional predictions, including phosphoesterase, could not be excluded. THEMATICS, a computational method for the prediction of active sites from protein 3D structures, identified potential reactive residues in YP_910028.1. Further analysis of the predicted active site and local comparison with its closest structure matches strongly suggested phosphoesterase activity, which was confirmed experimentally. Primer extension assays on both normal and mismatched DNA show neither extension nor degradation and provide evidence that YP_910028.1 has neither DNA polymerase activity nor DNA proofreading activity. These results suggest that many of the sequence neighbors previously annotated as having DNA polymerase activity may actually be misannotated.
Functional annotation; structural genomics; phosphoesterase; THEMATICS; active site prediction
DEN refinement and automated model building with AutoBuild were used to determine the structure of a putative succinyl-diaminopimelate desuccinylase from C. glutamicum. This difficult case of molecular-replacement phasing shows that the synergism between DEN refinement and AutoBuild outperforms standard refinement protocols.
Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
reciprocal-space refinement; DEN refinement; real-space refinement; automated model building; succinyl-diaminopimelate desuccinylase
Using the COG database, a comparative genome analysis from anaerobic and aerobic microorganisms, was performed with the aim of identifying proteins specific to the anaerobic way of life. Thirty-three COGs were identified, five of which corresponded to proteins of unknown function. We focused our study on TM0486, from Thermotoga maritima, that belongs to one of these latter COGs of unknown function, namely COG0011. The crystal structure of the protein was determined at 2 Å resolution. The structure adopts a βαββαβ ferredoxin-like fold and assembles as a homotetramer. The structure also revealed the presence of a pocket in each monomer that bound an unidentified ligand
NMR and calorimetric experiments revealed that TM0486 specifically bound thiamin with a Kd of 1.58 µM, but not hydroxymethyl pyrimidine (HMP), that was implicated previously as a potential ligand. We demonstrated that the TM0486 gene belongs to the same multicistronic unit as TM0483, TM0484 and TM0485. Although these three genes have already been assigned to the transport of HMP, with TM0484 being the periplasmic thiamin/HMP binding protein and TM0485 and TM0483 the transmembrane and the ATPase components, respectively, our results led us to conclude that this operon encodes for an ABC transporter dedicated to thiamin, with TM0486 transporting charged thiamin in the cytoplasm. Given that this transcriptional unit was up-regulated when T. maritima was exposed to oxidative conditions, we propose that by chelating cytoplasmic thiamin, TM0486 and, by extension, proteins belonging to COG0011 are involved in the response mechanism to stress that could arise during aerobic conditions.
Thermotoga maritima; Unknown function protein; DUF77; Thiamin binding protein; Oxidative stress
The software suite Xsolve semi-exhaustively explores key parameters of the X-ray structure-determination process to compute multiple three-dimensional protein structures independently and in parallel from a set of diffraction images. An optimal consensus model for subsequent manual refinement is computed from these structures.
The Joint Center for Structural Genomics (JCSG), one of four large-scale structure-determination centers funded by the US Protein Structure Initiative (PSI) through the National Institute for General Medical Sciences, has been operating an automated distributed structure-solution pipeline, Xsolve, for well over half a decade. During PSI-2, Xsolve solved, traced and partially refined 90% of the JCSG’s nearly 770 MAD/SAD structures at an average resolution of about 2 Å without human intervention. Xsolve executes many well established publicly available crystallography software programs in parallel on a commodity Linux cluster, resulting in multiple traces for any given target. Additional software programs have been developed and integrated into Xsolve to further minimize human effort in structure refinement. ConsensusModeler exploits complementarities in traces from Xsolve to compute a single optimal model for manual refinement. Xpleo is a powerful robotics-inspired algorithm to build missing fragments and qFit automatically identifies and fits alternate conformations.
distributed protein-structure determination; consensus models; parallel computing
CvfB is a conserved regulatory protein important for the virulence of Staphylococcus aureus. We show here that CvfB binds RNA. The crystal structure of the CvfB ortholog from Streptococcus pneumoniae at 1.4 Å resolution reveals a unique RNA binding protein that is formed from a concatenation of well-known structural modules that bind nucleic acids: three consecutive S1 RNA-binding domains and a winged-helix (WH) domain. The third S1 and the WH domains are required for cooperative RNA binding and form a continuous surface that likely contributes to the RNA interaction. The WH domain is critical to CvfB function and contains a unique structural motif. Thus CvfB represents a novel assembly of modules for binding RNA.
CvfB; Winged-helix domain; S1 domain; RNA binding; virulence
Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.
Cell Differentiation; Cellular Regulation; Myc; Tumor Suppressor; X-ray Crystallography; Apoptosis; NDRG Family; NDRG2
A new algorithm that automatically models discrete heterogeneity in X-ray data demonstrates that the variability observed at high resolution can be adequately represented by including correlated structural features in protein models. The algorithm is based on simultaneous exploration of a very large number of alternative interpretations of electron-density maps.
The native state of a protein is regarded to be an ensemble of conformers, which allows association with binding partners. While some of this structural heterogeneity is retained upon crystallization, reliably extracting heterogeneous features from diffraction data has remained a challenge. In this study, a new algorithm for the automatic modelling of discrete heterogeneity is presented. At high resolution, the authors’ single multi-conformer model, with correlated structural features to represent heterogeneity, shows improved agreement with the diffraction data compared with a single-conformer model. The model appears to be representative of the set of structures present in the crystal. In contrast, below 2 Å resolution representing ambiguous electron density by correlated multi-conformers in a single model does not yield better agreement with the experimental data. Consistent with previous studies, this suggests that variability in multi-conformer models at lower resolution levels reflects uncertainty more than coordinated motion.
heterogeneity; modeling; multi-conformers
The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years and has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe.
The Joint Center for Structural Genomics high-throughput structural biology pipeline has delivered more than 1000 structures to the community over the past ten years. The JCSG has made a significant contribution to the overall goal of the NIH Protein Structure Initiative (PSI) of expanding structural coverage of the protein universe, as well as making substantial inroads into structural coverage of an entire organism. Targets are processed through an extensive combination of bioinformatics and biophysical analyses to efficiently characterize and optimize each target prior to selection for structure determination. The pipeline uses parallel processing methods at almost every step in the process and can adapt to a wide range of protein targets from bacterial to human. The construction, expansion and optimization of the JCSG gene-to-structure pipeline over the years have resulted in many technological and methodological advances and developments. The vast number of targets and the enormous amounts of associated data processed through the multiple stages of the experimental pipeline required the development of variety of valuable resources that, wherever feasible, have been converted to free-access web-based tools and applications.
structural genomics; Joint Center for Structural Genomics; Protein Structure Initiative
As noticed by generations of structural biologists, closely homologous proteins may have substantially different crystallization properties and propensities. These observations can be used to systematically introduce additional dimensionality into crystallization trials by targeting homologous proteins from multiple genomes in a “genome pool” strategy. Through extensive use of our recently introduced “crystallization feasibility score” (Slabinski et al., 2007a), we can explain that the genome pool strategy works well because the crystallization feasibility scores are surprisingly broad within families of homologous proteins, with most families containing a range of optimal to very difficult targets. We also show that some families can be regarded as relatively “easy”, where a significant number of proteins are predicted to have optimal crystallization features, and others are “very difficult”, where almost none are predicted to result in a crystal structure. Thus, the outcome of such variable distributions of such crystallizability' preferences leads to uneven structural coverage of known families, with “easier” or “optimal” families having several times more solved structures than “very difficult” ones. Nevertheless, this latter category can be successfully targeted by increasing the number of genomes that are used to select targets from a given family. On average, adding 10 new genomes to the “genome pool” provides more promising targets for 7 “very difficult” families. In contrast, our crystallization feasibility score does not indicate that any specific microbial genomes can be readily classified as “easier” or “very difficult” with respect to providing suitable candidates for crystallization and structure determination. Finally, our analyses show that specific physicochemical properties of the protein sequence favor successful outcomes for structure determination and, hence, the group of proteins with known 3D structures is systematically different from the general pool of known proteins. We, therefore, assess the structural consequences of these differences in protein sequence and protein biophysical properties.
X-ray crystallography; protein crystallization; Protein Structure Initiative; structural genomics; target selection
Metabolic pathways have traditionally been described in terms of biochemical reactions and metabolites. Using structural genomics and systems biology, we generated a three-dimensional reconstruction of the central metabolic network of the bacterium, Thermotoga maritima (TM). The network encompassed 478 proteins of which 120 were determined by experiment and 358 were modeled. Structural analysis revealed that proteins forming the network are dominated by a small number (only 182) of basic shapes (folds) performing diverse, but mostly related functions. Most of these folds are already present in the essential core (~30%) of the network, and its expansion by nonessential proteins is achieved with relatively few additional folds. Thus, integration of structural data with networks analysis generates insight into the function, mechanism and evolution of biological networks.
Determination of first protein structures, from hundreds of families of unknown function, have shown that divergence, rather than novelty, is the dominant force that shapes the evolution of the protein universe.
The genome projects have unearthed an enormous diversity of genes of unknown function that are still awaiting biological and biochemical characterization. These genes, as most others, can be grouped into families based on sequence similarity. The PFAM database currently contains over 2,200 such families, referred to as domains of unknown function (DUF). In a coordinated effort, the four large-scale centers of the NIH Protein Structure Initiative have determined the first three-dimensional structures for more than 250 of these DUF families. Analysis of the first 248 reveals that about two thirds of the DUF families likely represent very divergent branches of already known and well-characterized families, which allows hypotheses to be formulated about their biological function. The remainder can be formally categorized as new folds, although about one third of these show significant substructure similarity to previously characterized folds. These results infer that, despite the enormous increase in the number and the diversity of new genes being uncovered, the fold space of the proteins they encode is gradually becoming saturated. The previously unexplored sectors of the protein universe appear to be primarily shaped by extreme diversification of known protein families, which then enables organisms to evolve new functions and adapt to particular niches and habitats. Notwithstanding, these DUF families still constitute the richest source for discovery of the remaining protein folds and topologies.
More than 40% of known proteins lack any annotation within public databases and are usually referred to as hypothetical proteins despite most of them being real and many being evolutionarily conserved and thus expected to play important biological roles. Determination of the three-dimensional structures of representatives of more than 240 families of protein domains of unknown function by the Protein Structure Initiative has provided a unique sample of regions of the protein universe that, until this systematic effort, were completely uncharacterized. Analysis of these structures reveals that most of the 240 families can be considered as remote homologs of already known protein families. Such distant evolutionary links can sometimes be predicted by current state-of-the-art sequence comparison tools, but structural analysis has led to the first hypotheses about biological functions for many of these uncharacterized proteins, and serves as a starting point for experimental studies. The rapid pace of discovery of such relationships appears to suggest that the protein universe is made up of a relatively small and stable number of ‘extended neighborhoods’ that bring together distantly related protein families. Thus, the vast uncharacterized part of protein universe, called by some “the dark matter of protein space”, may consist mainly of highly divergent homologs. Continued structural characterization of these previously under-investigated regions of the protein universe should further help unravel the patterns and rules that led to such divergence in the evolution of protein structure and function.
Crystallographic end-stations require a significant investment in state-of-the-art equipment, as well as a significant effort in software development. The equipment often sits idle during annual maintenance shutdowns. In order to utilize the existing hardware and software during these shutdowns, we installed a sealed-tube microsource X-ray generator in the beamline 9-2 hutch at Stanford Synchrotron Radiation Laboratory. A multi-layer optic provides good flux and spectral purity. The small physical size of the source, the long optic to focus distance (635 mm) and the short source to optic distance (65 mm) allowed the use of existing beamline components, without any significant modification. The system replaces a short section of beam pipe upstream of the beam conditioning slits and shutter. The system can be installed and removed from the beamline in less than 1 day.
The Joint Center for Structural Genomics (JCSG) and SSRL Structural Molecular Biology group developed the Stanford Automated Mounting (SAM) system and installed it on beamlines at SSRL. The JCSG relies on this system to test crystals for diffraction. The installation of the X-ray microsource in beamline 9-2 allowed crystal screening to continue during SSRL shutdowns. Using a standard screening protocol of two 10 minute exposures, separated by a 90° phi rotation, the system was capable of screening up to 400 crystals per week and was left to run unattended for up to 4 days. Over 8200 crystals were screened during the last four SSRL shutdown periods.
An X-ray generator can also be useful for ongoing beamline development. Shutdown periods provide easier access to the experimental hardware, however, some tests require beam. The X-ray microsource offers the ability to conduct these tests during periods when users are not scheduled.
Protein Crystallography; Crystal Screening; X-ray Generator
Bacterial spore formation is a complex process of fundamental relevance to biology and human disease. The spore coat structure is complex and poorly understood, and the roles of many of the protein components remain unclear. We describe a new family of spore coat proteins, the bacterial spore kinases (BSKs), and the first crystal structure of a BSK, YtaA (CotI) from Bacillus subtilis. BSKs are widely distributed in spore-forming Bacillus and Clostridium species, and have a dynamic evolutionary history. Sequence and structure analyses indicate that the BSKs are CAKs, a prevalent group of small molecule kinases in bacteria that is distantly related to the eukaryotic protein kinases. YtaA has substantial structural similarity to CAKs, but also displays distinctive features that broaden our understanding of the CAK group. Evolutionary constraint analysis of the protein surfaces indicates that members of the BSK family have distinct clade-conserved patterns in the substrate binding region, and probably bind and phosphorylate distinct targets. Several classes of BSKs have apparently independently lost catalytic activity to become pseudokinases, indicating that the family also has a major noncatalytic function. Proteins 2010. © 2009 Wiley-Liss, Inc.
protein kinase-like; PKL; CAK; endospore; pseudokinase; YtaA; CotS; YutH; YsxE; BSK
The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370–495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.
Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase (GGR) family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with FAD and a bacterial lipid. The DGGR structure can be assigned to the well-studied, para-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two narrow curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the structure and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of GGRs. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.
Imelysin-like proteins define a superfamily of bacterial proteins that are likely involved in iron uptake. Members of this superfamily were previously thought to be peptidases and were included in the MEROPS family M75. We determined the first crystal structures of two remotely related, imelysin-like proteins. The Psychrobacter arcticus structure was determined at 2.15 Å resolution and contains the canonical imelysin fold, while higher resolution structures from the gut bacteria Bacteroides ovatus, in two crystal forms (at 1.25 Å and 1.44 Å resolution), have a circularly permuted topology. Both structures are highly similar to each other despite low sequence similarity and circular permutation. The all-helical structure can be divided into two similar four-helix bundle domains. The overall structure and the GxHxxE motif region differ from known HxxE metallopeptidases, suggesting that imelysin-like proteins are not peptidases. A putative functional site is located at the domain interface. We have now organized the known homologous proteins into a superfamily, which can be separated into four families. These families share a similar functional site, but each has family-specific structural and sequence features. These results indicate that imelysin-like proteins have evolved from a common ancestor, and likely have a conserved function.
NlpC/P60 superfamily papain-like enzymes play important roles in all kingdoms of life. Two members of this superfamily, LRAT-like and YaeF/YiiX-like families, were predicted to contain a catalytic domain that is circularly permuted such that the catalytic cysteine is located near the C-terminus, instead of at the N-terminus. These permuted enzymes are widespread in virus, pathogenic bacteria, and eukaryotes. We determined the crystal structure of a member of the YaeF/YiiX-like family from Bacillus cereus in complex with lysine. The structure, which adopts a ligand-induced, “closed” conformation, confirms the circular permutation of catalytic residues. A comparative analysis of other related protein structures within the NlpC/P60 superfamily is presented. Permutated NlpC/P60 enzymes contain a similar conserved core and arrangement of catalytic residues, including a Cys/His-containing triad and an additional conserved tyrosine. More surprisingly, permuted enzymes have a hydrophobic S1 binding pocket that is distinct from previously characterized enzymes in the family, indicative of novel substrate specificity. Further analysis of a structural homolog, YiiX (PDB 2if6) identified a fatty acid in the conserved hydrophobic pocket, thus providing additional insights into possible function of these novel enzymes.
Bacterial cell walls contain peptidoglycan, an essential polymer made by enzymes in the Mur pathway. These proteins are specific to bacteria, which make them targets for drug discovery. MurC, MurD, MurE and MurF catalyze the synthesis of the peptidoglycan precursor UDP-N-acetylmuramoyl-L-alanyl-γ-D-glutamyl-meso-diaminopimelyl-D-alanyl-D-alanine by the sequential addition of amino acids onto UDP-N-acetylmuramic acid (UDP-MurNAc). MurC-F enzymes have been extensively studied by biochemistry and X-ray crystallography. In Gram-negative bacteria, ∼30–60% of the bacterial cell wall is recycled during each generation. Part of this recycling process involves the murein peptide ligase (Mpl), which attaches the breakdown product, the tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate, to UDP-MurNAc. We present the crystal structure at 1.65 Å resolution of a full-length Mpl from the permafrost bacterium Psychrobacter arcticus 273-4 (PaMpl). Although the Mpl structure has similarities to Mur enzymes, it has unique sequence and structure features that are likely related to its role in cell wall recycling, a function that differentiates it from the MurC-F enzymes. We have analyzed the sequence-structure relationships that are unique to Mpl proteins and compared them to MurC-F ligases. We have also characterized the biochemical properties of this enzyme (optimal temperature, pH and magnesium binding profiles and kinetic parameters). Although the structure does not contain any bound substrates, we have identified ∼30 residues that are likely to be important for recognition of the tripeptide and UDP-MurNAc substrates, as well as features that are unique to Psychrobacter Mpl proteins. These results provide the basis for future mutational studies for more extensive function characterization of the Mpl sequence-structure relationships.
The crystal structure of tryptophanyl-tRNA synthetase from T. maritima unexpectedly revealed an iron–sulfur cluster bound to the tRNA anticodon-binding region.
A novel aminoacyl-tRNA synthetase that contains an iron–sulfur cluster in the tRNA anticodon-binding region and efficiently charges tRNA with tryptophan has been found in Thermotoga maritima. The crystal structure of TmTrpRS (tryptophanyl-tRNA synthetase; TrpRS; EC 184.108.40.206) reveals an iron–sulfur [4Fe–4S] cluster bound to the tRNA anticodon-binding (TAB) domain and an l-tryptophan ligand in the active site. None of the other T. maritima aminoacyl-tRNA synthetases (AARSs) contain this [4Fe–4S] cluster-binding motif (C-x
2-C). It is speculated that the iron–sulfur cluster contributes to the stability of TmTrpRS and could play a role in the recognition of the anticodon.
TM0492; tryptophanyl-tRNA ligase; tryptophanyl-tRNA synthetase class I; iron–sulfur clusters; structural genomics
The crystal structure of the prephenate dehydrogenase component of the bifunctional H. influenzae TyrA reveals unique structural differences between bifunctional and monofunctional TyrA enzymes.
Chorismate mutase/prephenate dehydrogenase from Haemophilus influenzae Rd KW20 is a bifunctional enzyme that catalyzes the rearrangement of chorismate to prephenate and the NAD(P)+-dependent oxidative decarboxylation of prephenate to 4-hydroxyphenylpyruvate in tyrosine biosynthesis. The crystal structure of the prephenate dehydrogenase component (HinfPDH) of the TyrA protein from H. influenzae Rd KW20 in complex with the inhibitor tyrosine and cofactor NAD+ has been determined to 2.0 Å resolution. HinfPDH is a dimeric enzyme, with each monomer consisting of an N-terminal α/β dinucleotide-binding domain and a C-terminal α-helical dimerization domain. The structure reveals key active-site residues at the domain interface, including His200, Arg297 and Ser179 that are involved in catalysis and/or ligand binding and are highly conserved in TyrA proteins from all three kingdoms of life. Tyrosine is bound directly at the catalytic site, suggesting that it is a competitive inhibitor of HinfPDH. Comparisons with its structural homologues reveal important differences around the active site, including the absence of an α–β motif in HinfPDH that is present in other TyrA proteins, such as Synechocystis sp. arogenate dehydrogenase. Residues from this motif are involved in discrimination between NADP+ and NAD+. The loop between β5 and β6 in the N-terminal domain is much shorter in HinfPDH and an extra helix is present at the C-terminus. Furthermore, HinfPDH adopts a more closed conformation compared with TyrA proteins that do not have tyrosine bound. This conformational change brings the substrate, cofactor and active-site residues into close proximity for catalysis. An ionic network consisting of Arg297 (a key residue for tyrosine binding), a water molecule, Asp206 (from the loop between β5 and β6) and Arg365′ (from the additional C-terminal helix of the adjacent monomer) is observed that might be involved in gating the active site.
tyrosine biosynthesis; prephenate; chorismate; Haemophilus influenzae; structural genomics
Cell cycle regulated stalk biogenesis in Caulobacter crescentus is controlled by a multi-step phosphorelay system consisting of the hybrid histidine kinase ShkA, the histidine-phosphotransfer protein ShpA and the response regulator TacA. ShpA shuttles phosphoryl groups between ShkA and TacA. When phosphorylated, TacA triggers a downstream transcription cascade for stalk synthesis in an RpoN-dependent manner. The crystal structure of ShpA was determined to 1.52 Å resolution. ShpA belongs to a family of monomeric histidine phosphotransfer (HPt) proteins, which feature a highly conserved four-helix bundle. The phosphorylatable histidine, His56, is located on the surface of the helix bundle and is fully solvent exposed. One end of the four-helix bundle in ShpA is shorter compared to other characterized histidine phosphotransfer proteins, whereas the face that potentially interacts with the response regulators is structurally conserved. Similarities of the interaction surface around the phosphorylation site suggest that ShpA is likely to share a common mechanism for molecular recognition and phosphotransfer with yeast phosphotransfer protein YPD1 despite low overall sequence similarity.
Stalk biogenesis; phosphorelay; two-component signal transduction; Histidine phosphotransfer protein (HPt)