Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases.

beta-Lactamases are the resistance enzymes for beta-lactam antibiotics, of which four classes are known. beta-lactamases hydrolyze the beta-lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 beta-lactamase depends critically on an unusual carbamylated lysine as the basic residue for both the enzyme acylation and deacylation steps of catalysis. The formation of carbamylated lysine is reversible. Evidence is presented that this enzyme is dimeric and carbamylated in living bacteria. High-resolution x-ray structures for the native enzyme were determined at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are present per asymmetric unit. One monomer in each dimer was carbamylated at pH 6.0, whereas all four monomers were fully carbamylated at pH 8.5. At the intermediate pH values, one monomer of each dimer was carbamylated, and the other showed a mixture of carbamylated and non-carbamylated lysines. It would appear that, as the pH increased for the sample, additional lysines were "titrated" by carbamylation. A handful of carbamylated lysines are known from protein crystallographic data, all of which have been attributed roles in structural stabilization (mostly as metal ligands) of the proteins. This paper reports a previously unrecognized role for a noncoordinated carbamylate lysine as a basic residue involved in mechanistic reactions of an enzyme, which indicates another means for expansion of the catalytic capabilities of the amino acids in nature beyond the 20 common amino acids in development of biological catalysts.

Crystal structure of the CheA histidine phosphotransfer domain that mediates response regulator phosphorylation in bacterial chemotaxis.

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.

Further insights into the mechanism of function of the response regulator CheY from crystallographic studies of the CheY--CheA(124--257) complex.

New crystallographic structures of the response regulator CheY in association with CheA(124--257), its binding domain in the kinase CheA, have been determined. In all crystal forms, the molecular interactions at the heterodimer interface are identical. Soaking experiments have been performed on the crystals using acetyl phosphate as phosphodonor to CheY. No phosphoryl group attached to Asp57 of CheY is visible from the electron density, but the response regulator in the CheY-CheA(124--257) complex may have undergone a phosphorylation-dephosphorylation process. The distribution of water molecules and the geometry of the active site have changed and are now similar to those of isolated CheY. In a second soaking experiment, imido-diphosphate, an inhibitor of the phosphorylation reaction, was used. This compound binds in the vicinity of the active site, close to the N-terminal part of the first alpha-helix. Together, these results suggest that the binding of CheY to CheA(124--257) generates a geometry of the active site that favours phosphorylation and that imido-diphosphate interferes with phosphorylation by precluding structural changes in this region.

Crystal structure of the F component of the Panton-Valentine leucocidin.

Leucocidins and gamma-hemolysins are bi-component staphylococcal toxins that form lytic transmembrane pores. Their cytotoxic activities involve the synergistic association of a class S and a class F component, produced as water-soluble monomers which assemble on the surface of specific cells. The structure of the F protein from Panton-Valentine leucocidin, solved at 2.0 A resolution, and sequence alignment suggest that it represents the fold of any secreted protein in this family of toxins. The comparison of this structure to that of the homoheptameric alpha-hemolysin provides some insights into the molecular events that may occur during pore formation.

The high resolution crystal structure for class A beta-lactamase PER-1 reveals the bases for its increase in breadth of activity.

The treatment of infectious diseases by beta-lactam antibiotics is continuously challenged by the emergence and dissemination of new beta-lactamases. In most cases, the cephalosporinase activity of class A enzymes results from a few mutations in the TEM and SHV penicillinases. The PER-1 beta-lactamase was characterized as a class A enzyme displaying a cephalosporinase activity. This activity was, however, insensitive to the mutations of residues known to be critical for providing extended substrate profiles to TEM and SHV. The x-ray structure of the protein, solved at 1.9-A resolution, reveals that two of the most conserved features in class A beta-lactamases are not present in this enzyme: the fold of the Omega-loop and the cis conformation of the peptide bond between residues 166 and 167. The new fold of the Omega-loop and the insertion of four residues at the edge of strand S3 generate a broad cavity that may easily accommodate the bulky substituents of cephalosporin substrates. The trans conformation of the 166-167 bond is related to the presence of an aspartic acid at position 136. Selection of class A enzymes based on the occurrence of both Asp(136) and Asn(179) identifies a subgroup of enzymes with high sequence homology.

Insights into class D beta-lactamases are revealed by the crystal structure of the OXA10 enzyme from Pseudomonas aeruginosa.

BACKGROUND: beta-lactam antibiotic therapies are commonly challenged by the hydrolytic activities of beta-lactamases in bacteria. These enzymes have been grouped into four classes: A, B, C, and D. Class B beta-lactamases are zinc dependent, and enzymes of classes A, C, and D are transiently acylated on a serine residue in the course of the turnover chemistry. While class A and C beta-lactamases have been extensively characterized by biochemical and structural methods, class D enzymes remain the least studied despite their increasing importance in the clinic. RESULTS: The crystal structure of the OXA10 class D beta-lactamase has been solved to 1.66 A resolution from a gold derivative and MAD phasing. This structure reveals that beta-lactamases from classes D and A, despite very poor sequence similarity, share a similar overall fold. An additional beta strand in OXA10 mediates the association into dimers characterized by analytical ultracentrifugation. Major differences are found when comparing the molecular details of the active site of this class D enzyme to the corresponding regions in class A and C beta-lactamases. In the native structure of the OXA10 enzyme solved to 1.8 A, Lys-70 is carbamylated. CONCLUSIONS: Several features were revealed by this study: the dimeric structure of the OXA10 beta-lactamase, an extension of the substrate binding site which suggests that class D enzymes may bind other substrates beside beta-lactams, and carbamylation of the active site Lys-70 residue. The CO2-dependent activity of the OXA10 enzyme and the kinetic properties of the natural OXA17 mutant protein suggest possible relationships between carbamylation, inhibition of the enzyme by anions, and biphasic behavior of the enzyme.

6-(hydroxyalkyl)penicillanates as probes for mechanisms of beta-lactamases.

6-(Hydroxyalkyl)penicillanates have proven helpful as probes for the mechanisms of beta-lactamases, enzymes of resistance for beta-lactam antibiotics. The present report summarizes the concepts on design, syntheses and use of these molecules in mechanistic studies of beta-lactamases.

Discoupling the Ca(2+)-activation from the pore-forming function of the bi-component Panton-Valentine leucocidin in human PMNs.

The consecutive cell activation, including Ca(2+)-channel opening, and pore formation leading to human neutrophil lysis were the two functions of the staphylococcal Panton-Valentine leucocidin attempted to be discoupled by site-directed mutagenesis. In a first approach consisting in deletions of the cytoplasmic extremity of the transmembranous domain, we produced a LukF-PV DeltaSer125-Leu128 with a slightly reduced Ca(2+) induction but with a significantly lowered lytic activity when combined with its synergistic protein LukS-PV. The second approach consisted in the modification of charges and/or introduction of a steric hindrance inside the pore, which also led to interesting mutated proteins: LukF-PV G131D, G131W and G130D. The latter had an intact Ca(2+) induction ability while the lytic one was 20-fold diminished. Binding properties and intrinsic pore diameters of these discoupled toxins remained comparable to the wild-type protein. The mutated proteins promoted interleukin-8 secretion, but they were rather inactive in an experimental model. New insights are brought concerning the role of the two functions in the virulence of this bi-component leucotoxin.

Inhibition of the broad spectrum nonmetallocarbapenamase of class A (NMC-A) beta-lactamase from Enterobacter cloacae by monocyclic beta-lactams.

beta-Lactamases hydrolyze beta-lactam antibiotics, a reaction that destroys their antibacterial activity. These enzymes, of which four classes are known, are the primary cause of resistance to beta-lactam antibiotics. The class A beta-lactamases form the largest group. A novel class A beta-lactamase, named the nonmetallocarbapenamase of class A (NMC-A) beta-lactamase, has been discovered recently that has a broad substrate profile that included carbapenem antibiotics. This is a serious development, since carbapenems have been relatively immune to the action of these resistance enzymes. Inhibitors for this enzyme are sought. We describe herein that a type of monobactam molecule of our design inactivates the NMC-A beta-lactamase rapidly, efficiently, and irreversibly. The mechanism of inactivation was investigated by solving the x-ray structure of the inhibited NMC-A enzyme to 1.95 A resolution. The structure shed light on the nature of the fragmentation of the inhibitor on enzyme acylation and indicated that there are two acyl-enzyme species that account for enzyme inhibition. Each of these inhibited enzyme species is trapped in a distinct local energy minimum that does not predispose the inhibitor species for deacylation, accounting for the irreversible mode of enzyme inhibition. Molecular dynamics simulations provided evidence in favor of a dynamic motion for the acyl-enzyme species, which samples a considerable conformational space prior to the entrapment of the two stable acyl-enzyme species in the local energy minima. A discussion of the likelihood of such dynamic motion for turnover of substrates during the normal catalytic processes of the enzyme is presented.

X-ray structure of the Asn276Asp variant of the Escherichia coli TEM-1 beta-lactamase: direct observation of electrostatic modulation in resistance to inactivation by clavulanic acid.

The clinical use of beta-lactam antibiotics combined with beta-lactamase inactivators, such as clavulanate, has resulted in selection of beta-lactamases that are insensitive to inactivation by these molecules. Therefore, therapeutic combinations of an enzyme inactivator and a penicillin are harmless for bacteria harboring such an enzyme. The TEM beta-lactamase variants are the most frequently encountered enzymes of this type, and presently, 20 variants are designated as inhibitor-resistant TEM ("IRT") enzymes. Three mutations appear to account for the phenotype of the majority of IRT enzymes, one of them being the Asn276Asp substitution. In this study, we have characterized the kinetic properties of the inhibition process of the wild-type TEM-1 beta-lactamase and of its Asn276Asp variant with the three clinically used inactivators, clavulanic acid (clavulanate), sulbactam, and tazobactam, and we report the X-ray structure for the mutant variant at 2.3 A resolution. The changes in kinetic parameters for the interactions of the inhibitors with the wild-type and the mutant enzymes were more pronounced for clavulanate, and relatively inconsequential for sulbactam and tazobactam. The structure of the Asn276Asp mutant enzyme revealed a significant movement of Asp276 and the formation of a salt bridge of its side chain with the guanidinium group of Arg244, the counterion of the inhibitor carboxylate. A water molecule critical for the inactivation chemistry by clavulanate, which is observed in the wild-type enzyme structure, is not present in the crystal structure of the mutant variant. Such structural changes favor the turnover process over the inactivation chemistry for clavulanate, with profound phenotypic consequences. The report herein represents the best studied example of inhibitor-resistant beta-lactamases.

The structure of a Staphylococcus aureus leucocidin component (LukF-PV) reveals the fold of the water-soluble species of a family of transmembrane pore-forming toxins.

BACKGROUND: Leucocidins and gamma-hemolysins are bi-component toxins secreted by Staphylococcus aureus. These toxins activate responses of specific cells and form lethal transmembrane pores. Their leucotoxic and hemolytic activities involve the sequential binding and the synergistic association of a class S and a class F component, which form hetero-oligomeric complexes. The components of each protein class are produced as non-associated, water-soluble proteins that undergo conformational changes and oligomerization after recognition of their cell targets. RESULTS: The crystal structure of the monomeric water-soluble form of the F component of Panton-Valentine leucocidin (LukF-PV) has been solved by the multiwavelength anomalous dispersion (MAD) method and refined at 2.0 A resolution. The core of this three-domain protein is similar to that of alpha-hemolysin, but significant differences occur in regions that may be involved in the mechanism of pore formation. The glycine-rich stem, which undergoes a major rearrangement in this process, forms an additional domain in LukF-PV. The fold of this domain is similar to that of the neurotoxins and cardiotoxins from snake venom. CONCLUSIONS: The structure analysis and a multiple sequence alignment of all toxic components, suggest that LukF-PV represents the fold of any water-soluble secreted protein in this family of transmembrane pore-forming toxins. The comparison of the structures of LukF-PV and alpha-hemolysin provides some insights into the mechanism of transmembrane pore formation for the bi-component toxins, which may diverge from that of the alpha-hemolysin heptamer.

Conformational changes induced by phosphorylation of the FixJ receiver domain.

BACKGROUND: A variety of bacterial adaptative cellular responses to environmental stimuli are mediated by two-component signal transduction pathways. In these phosphorelay cascades, histidine kinases transphosphorylate a conserved aspartate in the receiver domain, a conserved module in the response regulator superfamily. The main effect of this phosphorylation is to alter the conformation of the response regulator in order to modulate its biological function. The response regulator FixJ displays a typical modular arrangement, with a phosphorylatable N-terminal receiver domain and a C-terminal DNA-binding domain. In the symbiotic bacterium Sinorhizobium meliloti, phosphorylation of this response regulator activates transcription of nitrogen-fixation genes. RESULTS: The crystal structures of the phosphorylated and of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) were solved at 2.3 A and 2.4 A resolution, respectively. They reveal the environment of the phosphoaspartate in the active site and the specific conformational changes leading to activation of the response regulator. Phosphorylation of the conserved aspartate induces major structural changes in the beta 4-alpha 4 loop, and in the signaling surface alpha 4-beta 5 that mediates dimerization of the phosphorylated full-length response regulator. A site-directed mutant at this protein-protein interface decreases the affinity of the phosphorylated response regulator for the fixK promoter tenfold. CONCLUSIONS: The cascade of phosphorylation-induced conformational changes in FixJN illustrates the role of conserved residues in stabilizing the phosphoryl group in the active site, triggering the structural transition and achieving the post-phosphorylation signaling events. We propose that these phosphorylation-induced conformational changes underly the activation of response regulators in general.

Structural transitions in the FixJ receiver domain.

BACKGROUND: Two-component signal transduction pathways are sophisticated phosphorelay cascades widespread in prokaryotes and also found in fungi, molds and plants. FixL/FixJ is a prototypical system responsible for the regulation of nitrogen fixation in the symbiotic bacterium Sinorhizobium meliloti. In microaerobic conditions the membrane-bound kinase FixL uses ATP to transphosphorylate a histidine residue, and the response regulator FixJ transfers the phosphoryl group from the phosphohistidine to one of its own aspartate residues in a Mg(2+)-dependent mechanism. RESULTS: Seven X-ray structures of the unphosphorylated N-terminal receiver domain of FixJ (FixJN) have been solved from two crystal forms soaked in different conditions. Three conformations of the protein were found. In the first case, the protein fold impairs metal binding in the active site and the structure reveals a receiver domain that is self-inhibited for catalysis. In the second conformation, the canonical geometry of the active site is attained, and subsequent metal binding to the protein induces minimal conformational changes. The third conformation illustrates a non-catalytic form of the protein where unwinding of the N terminus of helix alpha 1 has occurred. Interconversion of the canonical and self-inhibited conformations requires a large conformational change of the beta 3-alpha 3 loop region. CONCLUSIONS: These unphosphorylated structures of FixJN stress the importance of flexible peptide segments that delineate the active site. Their movements may act as molecular switches that define the functional status of the protein. Such observations are in line with structural and biochemical results obtained on other response regulator proteins and may illustrate general features that account for the specificity of protein-protein interactions.

X-ray analysis of the NMC-A beta-lactamase at 1.64-A resolution, a class A carbapenemase with broad substrate specificity.

The treatment of infectious diseases by penicillin and cephalosporin antibiotics is continuously challenged by the emergence and the dissemination of the numerous TEM and SHV mutant beta-lactamases with extended substrate profiles. These class A beta-lactamases nevertheless remain inefficient against carbapenems, the most effective antibiotics against clinically relevant pathogens. A new member of this enzyme class, NMC-A, was recently reported to hydrolyze at high rates, and hence destroy, all known beta-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-A resolution, and reveals modifications in the topology of the substrate-binding site. While preserving the geometry of the essential catalytic residues, the active site of the enzyme presents a disulfide bridge between residues 69 and 238, and certain other structural differences compared with the other beta-lactamases. These unusual features in class A beta-lactamases involve amino acids that participate in enzyme-substrate interactions, which suggested that these structural factors should be related to the very broad substrate specificity of this enzyme. The comparison of the NMC-A structure with those of other class A enzymes and enzyme-ligand complexes, indicated that the position of Asn-132 in NMC-A provides critical additional space in the region of the protein where the poorer substrates for class A beta-lactamases, such as cephamycins and carbapenems, need to be accommodated.

Crystal structure of the arcelin-1 dimer from Phaseolus vulgaris at 1.9-A resolution.

Arcelin-1 is a glycoprotein from kidney beans (Phaseolus vulgaris) which displays insecticidal properties and protects the seeds from predation by larvae of various bruchids. This lectin-like protein is devoid of monosaccharide binding properties and belongs to the phytohemagglutinin protein family. The x-ray structure determination at 1.9-A resolution of native arcelin-1 dimers, which correspond to the functional state of the protein in solution, was solved using multiple isomorphous replacement and refined to a crystallographic R factor of 0.208. The three glycosylation sites on each monomer are all covalently modified. One of these oligosaccharide chains provides interactions with protein atoms at the dimer interface, and another one may act by preventing the formation of higher oligomeric species in the arcelin variants. The dimeric structure and the severe alteration of the monosaccharide binding site in arcelin-1 correlate with the hemagglutinating properties of the protein, which are unaffected by simple sugars and sugar derivatives. Sequence analysis and structure comparisons of arcelin-1 with the other insecticidal proteins from kidney beans, arcelin-5, and alpha-amylase inhibitor and with legume lectins, yield insights into the molecular basis of the different biological functions of these proteins.

Oligomeric structure of the repressor of the bacteriophage Mu early operon.

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120-140 kDa at low concentrations, i.e. in the 10-microM range. This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.

Crystal structure of an acylation transition-state analog of the TEM-1 beta-lactamase. Mechanistic implications for class A beta-lactamases.

The crystal structure of a phosphonate complex of the class A TEM-1 beta-lactamase has been determined to a resolution of 2.0 A. The phosphonate appears stoichiometrically at the active site, bound covalently to Ser70Ogamma, with one phosphonyl oxygen in the oxyanion hole. Although the overall structure is very similar to that of the native enzyme (rms difference 0.37 A for all heavy atoms), changes have occurred in the position of active site functional groups. The active site is also not in the conformation observed in the complex of another class A beta-lactamase, that of Staphylococcus aureus PC1, with the same phosphonate [Chen, C. C. H., et al. (1993) J. Mol. Biol. 234,165-178]. Both phosphonate structures, however, can be seen to represent models of acylation transition-states since in each the deacylating water molecule appears firmly bound to the Glu166 carboxylate group. The major difference between the structures lies in the positioning of Lys73Nzeta and Ser130Ogamma. In the S. aureus structure, the closest interaction of these functional groups is between Lys73Nzeta and Ser70Ogamma (2.8 A), while in the TEM-1 structure it is between Ser130Ogamma and the second phosphonyl oxygen of the bound inhibitor (2.8 A). The former structure therefore may resemble a transition state for formation of the tetrahedral species in acylation by nucleophilic attack on the substrate, where Lys73Nzeta presumably catalyzes the reaction as a general base. The TEM-1 structure can then be seen as an analogue of the transition state for breakdown of the tetrahedral species, where Ser130Ogamma is acting as a general acid, assisting the departure of the leaving group. The class A beta-lactamase crystal structures now available lead to a self-consistent proposal for a mechanism of catalysis by these enzymes.

Characterization and sugar-binding properties of arcelin-1, an insecticidal lectin-like protein isolated from kidney bean (Phaseolus vulgaris L. cv. RAZ-2) seeds.

Arcelin-1 is a lectin-like protein found in the seeds of wild varieties of the kidney bean (Phaseolus vulgaris). This protein displays insecticidal properties, but the mechanism of action is as yet unknown. In the present study we investigated the biochemical and biophysical properties of arcelin-1 from Phaseolus vulgaris cv. RAZ-2. Native arcelin-1 is a dimeric glycoprotein of 60 kDa, built from the non-covalent association of two identical monomers. This dimer resists dissociation by chaotropic agents and is highly resistant to proteolytic enzymes. Each subunit contains 10% (w/w) neutral sugars which belong to the high-mannose and complex-type glycans attached to three glycosylation sites. No interaction of the protein with simple sugars could be detected, but arcelin-1 displays an intrinsic specificity in binding complex glycans. Arcelin-1 therefore differs from the closely related phytohaemagglutinin lectins and alpha-amylase inhibitor in several respects: oligomerization states, sugar-binding affinities and the type and number of glycan chains. These features may be related to the toxicity of arcelin-1.

Structure of the CheY-binding domain of histidine kinase CheA in complex with CheY.

Bacterial adaptation to the environment is accomplished through the coordinated activation of specific sensory receptors and signal processing proteins. Among the best characterized of these pathways are those which employ the two-component paradigm. In these systems, signal transmission is mediated by Mg(2+)-dependent phospho-relay reactions between histidine auto-kinases and phospho-accepting receiver domains in response-regulator proteins. Although this mechanism of activation is common to all response-regulators, detrimental cross-talk between different two-component pathways within the same cell is minimized through the use of specific recognition domains. Here, we report the crystal structure, at 2.95 A resolution, of the response regulator of bacterial chemotaxis, CheY, bound to the recognition domain from its cognate histidine kinase, CheA. The structure suggests that molecular recognition, in this low affinity complex (KD = 2 microM), may also contribute to the mechanism of CheY activation.

Small-angle X-ray scattering and crystallographic studies of arcelin-1: an insecticidal lectin-like glycoprotein from Phaseolus vulgaris L.

Arcelin-1 and alpha-amylase inhibitor are two lectin-like glycoproteins expressed in the seeds of the kidney bean (Phaseolus vulgaris). They display insecticidal activities and protect the seeds from predation by larvae of various bruchids through different biological actions. Solution-state investigations by small-angle X-ray scattering (SAXS) show the dimeric structure of arcelin-1, a requirement for its hemagglutinating properties. Anions were found to have specific properties in their effectiveness to disrupt protein aggregates, affect solubility, and improve crystallizability. The SAXS results were used to improve crystallization conditions, and single crystals diffracting beyond 1.9 A resolution were obtained. X-ray diffraction data analysis shows that noncrystallographic symmetry-related arcelin-1 molecules form a lectin-like dimer and reveals the presence of a solvent-exposed anion binding site on the protein, at a crystal-packing interface. The solution state properties of arcelin-1 and crystal twinning may be explained by the anion specificity of this binding site.