Crystal structure of glucansucrase from the dental caries pathogen Streptococcus mutans

Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite − 1. On the other hand, novel structural features were revealed in subsites + 1 and + 2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite + 1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.     

Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli

Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.     

Crystal structure of the C-terminal domain of a flagellar hook-capping protein from Xanthomonas campestris

The crystal structure of the C-terminal domain of a hook-capping protein FlgD from the plant pathogen Xanthomonas campestris (Xc) has been determined to a resolution of ca 2.5 Å using X-ray crystallography. The monomer of whole FlgD comprises 221 amino acids with a molecular mass of 22.7 kDa, but the flexible N-terminus is cleaved for up to 75 residues during crystallization. The final structure of the C-terminal domain reveals a novel hybrid comprising a tudor-like domain interdigitated with a fibronectin type III domain. The C-terminal domain of XcFlgD forms three types of dimers in the crystal. In agreement with this, analytical ultracentrifugation and gel filtration experiments reveal that they form a stable dimer in solution. From these results, we propose that the Xc flagellar hook cap protein FlgD comprises two individual domains, a flexible N-terminal domain that cannot be detected in the current study and a stable C-terminal domain that forms a stable dimer.     

Crystal structure of scytalidoglutamic peptidase with its first potent inhibitor provides insights into substrate specificity and catalysis

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the beta-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper beta-sheet and enables delineation of the S4 to S3' substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71-Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme-inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141-Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme-inhibitor interactions and mechanistic interpretations are discussed.     

An Iron–Sulfur Cluster in the Polymerase Domain of Yeast DNA Polymerase ε

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits—Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1–1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2^ins) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.     

Crystal Structure of a Full-Length Autotransporter

The autotransporter (AT) secretion mechanism is the most common mechanism for the secretion of virulence factors across the outer membrane (OM) from pathogenic Gram-negative bacteria. In addition, ATs have attracted biotechnological and biomedical interest for protein display on bacterial cell surfaces. Despite their importance, the mechanism by which passenger domains of ATs pass the OM is still unclear. The classical view is that the β-barrel domain provides the conduit through which the unfolded passenger moves, with the energy provided by vectorial folding of the β-strand-rich passenger on the extracellular side of the OM. We present here the first structure of a full-length AT, the esterase EstA from Pseudomonas aeruginosa, at a resolution of 2.5 Å. EstA has a relatively narrow, 12-stranded β-barrel that is covalently attached to the passenger domain via a long, curved helix that occupies the lumen of the β-barrel. The passenger has a structure that is dramatically different from that of other known passengers, with a globular fold that is dominated by α-helices and loops. The arrangement of secondary-structure elements suggests that the passenger can fold sequentially, providing the driving force for passenger translocation. The esterase active-site residues are located at the apical surface of the passenger, at the entrance of a large hydrophobic pocket that contains a bound detergent molecule that likely mimics substrate. The EstA structure provides insight into AT mechanism and will facilitate the design of fusion proteins for cell surface display.     

Crystal structures of penicillin-binding protein 3 from Pseudomonas aeruginosa: comparison of native and antibiotic-bound forms

We report the first crystal structures of a penicillin-binding protein (PBP), PBP3, from Pseudomonas aeruginosa in native form and covalently linked to two important β-lactam antibiotics, carbenicillin and ceftazidime. Overall, the structures of apo and acyl complexes are very similar; however, variations in the orientation of the amino-terminal membrane-proximal domain relative to that of the carboxy-terminal transpeptidase domain indicate interdomain flexibility. Binding of either carbenicillin or ceftazidime to purified PBP3 increases the thermostability of the enzyme significantly and is associated with local conformational changes, which lead to a narrowing of the substrate-binding cleft. The orientations of the two β-lactams in the active site and the key interactions formed between the ligands and PBP3 are similar despite differences in the two drugs, indicating a degree of flexibility in the binding site. The conserved binding mode of β-lactam-based inhibitors appears to extend to other PBPs, as suggested by a comparison of the PBP3/ceftazidime complex and the Escherichia coli PBP1b/ceftoxamine complex. Since P. aeruginosa is an important human pathogen, the structural data reveal the mode of action of the frontline antibiotic ceftazidime at the molecular level. Improved drugs to combat infections by P. aeruginosa and related Gram-negative bacteria are sought and our study provides templates to assist that process and allows us to discuss new ways of inhibiting PBPs.     

Energetics-based protein profiling on a proteomic scale: identification of proteins resistant to proteolysis

Native states of proteins are flexible, populating more than just the unique native conformation. The energetics and dynamics resulting from this conformational ensemble are inherently linked to protein function and regulation. Proteolytic susceptibility is one feature determined by this conformational energy landscape. As an attempt to investigate energetics of proteins on a proteomic scale, we challenged the Escherichia coli proteome with extensive proteolysis and determined which proteins, if any, have optimized their energy landscape for resistance to proteolysis. To our surprise, multiple soluble proteins survived the challenge. Maltose binding protein, a survivor from thermolysin digestion, was characterized by in vitro biophysical studies to identify the physical origin of proteolytic resistance. This experimental characterization shows that kinetic stability is responsible for the unusual resistance in maltose binding protein. The biochemical functions of the identified survivors suggest that many of these proteins may have evolved extreme proteolytic resistance because of their critical roles under stressed conditions. Our results suggest that under functional selection proteins can evolve extreme proteolysis resistance by modulating their conformational energy landscapes without the need to invent new folds, and that proteins can be profiled on a proteomic scale according to their energetic properties by using proteolysis as a structural probe.     

The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.     

Structural Basis of Conserved Cysteine in the Fibroblast Growth Factor Family: Evidence for a Vestigial Half-Cystine

The 22 members of the mouse/human fibroblast growth factor (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-19, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in ∼ 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.     

Dynamics may significantly influence the estimation of interatomic distances in biomolecular X-ray structures

Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥ 0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.     

From the periplasmic signaling domain to the extracellular face of an outer membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine outer membrane receptor

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB-ExbB-ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 A resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB-ExbB-ExbD involves the periplasmic domain, which displays a beta-alpha-beta fold composed of two alpha-helices sandwiched by two beta-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.     

Crystal Structures and Enzyme Mechanisms of a Dual Fucose Mutarotase/Ribose Pyranase

Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to l-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward l-fucose but also for the pyranase activity toward d-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.     

Determination of the transition state ensemble for the folding of ubiquitin from a combination of Phi and Psi analyses

Protein engineering techniques have emerged as powerful tools for characterizing transition states (TSs) for protein folding. Recently, the Ψ analysis, in which double-histidine mutations create the possibility of reversible crosslinking in the native state, has been proposed as an additional approach to the well-established Φ analysis. We present here a combination of these two procedures for defining the structure of the TS of ubiquitin, a small α/β protein that has been used extensively as a model system for both experimental and computational studies of the protein-folding process. We performed a series of molecular dynamics simulations in which Φ and Ψ values were used as ensemble-averaged structural restraints to determine an ensemble of structures representing the TS of ubiquitin. Although the available Ψ values for ubiquitin did not, by themselves, generate well-defined TS ensembles, the inclusion of the restricted set of zero or unity values, but not fractional ones, provided useful complementary information to the Φ analysis. Our results show that the TS of ubiquitin is formed by a relatively narrow ensemble of structures exhibiting an overall native-like topology in which the N-terminal and C-terminal regions are in close proximity.     

Using Soft X-Rays for a Detailed Picture of Divalent Metal Binding in the Nucleosome

Divalent metals associate with DNA in a site-selective manner, which can influence nucleosome positioning, mobility, compaction, and recognition by nuclear factors. We previously characterized divalent metal binding in the nucleosome core using hard (short-wavelength) X-rays allowing high-resolution crystallographic determination of the strongest affinity sites, which revealed that Mn²⁺ associates with the DNA major groove in a sequence- and conformation-dependent manner. In this study, we obtained diffraction data with soft X-rays at the Mn²⁺ absorption edge for a core particle crystal in the presence of 10 mM MnSO₄, mimicking prevailing Mg²⁺ concentration in the nucleus. This provides an exceptional view of counterion binding in the nucleosome through identification of 45 divalent metal binding sites.In addition to that at the well-characterized major interparticle interface, only one other histone-divalent metal binding site is found, which corresponds to a symmetry-related counterpart on the ‘free’ H2B α1 helix C-terminus. This emphasizes the importance of the α-helix dipole in ion binding and suggests that the H2B motif may serve as a nucleation site in nucleosome compaction. The 43 sites associated with the DNA are characterized by (1) high-affinity direct coordination at the most electrostatically favorable major groove locations, (2) metal hydrate binding to the major groove, (3) direct coordination to phosphate groups at sites of high charge density, (4) metal hydrate binding in the minor groove, or (5) metal hydrate-divalent anion pairing. Metal hydrates are found within the minor groove only at locations displaying a narrow range of high-intermediate width and to which histone N-terminal tails are not associated or proximal. This indicates that divalent metals and histone tails can both collaborate and compete in minor groove association, which sheds light on nucleosome solubility and chromatin compaction behavior.     

The Zn2 position in metallo-beta-lactamases is critical for activity: a study on chimeric metal sites on a conserved protein scaffold

Metallo-beta-lactamases (MbetaLs) are bacterial Zn(II)-dependent hydrolases that confer broad-spectrum resistance to beta-lactam antibiotics. These enzymes can be subdivided into three subclasses (B1, B2 and B3) that differ in their metal binding sites and their characteristic tertiary structure. To date there are no clinically useful pan-MbetaL inhibitors available, mainly due to the unawareness of key catalytic features common to all MbetaL brands. Here we have designed, expressed and characterized two double mutants of BcII, a di-Zn(II) B1-MbetaL from Bacillus cereus, namely BcII-R121H/C221D (BcII-HD) and BcII-R121H/C221S (BcII-HS). These mutants display modified environments at the so-called Zn2 site or DCH site, reproducing the metal coordination environments of structurally related metallohydrolases. Through a combination of structural and functional studies, we found that BcII-HD is an impaired beta-lactamase even as a di-Zn(II) enzyme, whereas BcII-HS exhibits the ability to exist as mono or di-Zn(II) species in solution, with different catalytic performances. We show that these effects result from an altered position of Zn2, which is incapable of providing a productive interaction with the substrate beta-lactam ring. These results indicate that the position of Zn2 is essential for a productive substrate binding and hydrolysis.     

Structure of bovine milk calcium phosphate determined by x-ray absorption spectroscopy

Calcium in cow's milk is mainly in the form of calcium phosphate-phosphoprotein complexes known as casein micelles. These micelles, in contrast to other phosphoprotein complexes in bone and other tissues, can be readily isolated and studied, but conventional techniques have given ambiguous and conflicting evidence on the structure of milk calcium phosphate. Extended X-ray absorption fine structure and near-edge structure measurements at the newly commissioned Synchrotron Radiation Source at Daresbury indicate that it closely resembles brushite, CaHPO₄·2H₂O. This result, and chemical analysis, requires that phosphate groups from the matrix phosphoproteins be incorporated in the brushite lattice, probably in the surface, suggesting that these organic phosphate groups act as heterogeneous nucleation sites for phase separation of the calcium phosphate from solution.