Jean-Pierre Samama

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.

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

β-Lactamases are the resistance enzymes for β-lactam antibiotics, of which four classes are known. β-lactamases hydrolyze the β-lactam moieties of these antibiotics, rendering them inactive. It is shown herein that the class D OXA-10 β-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.

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.

Insights into Class D β-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.

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 β-lactamases with extended substrate profiles. These class A β-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 β-lactam antibiotics, including carbapenems and cephamycins. The crystal structure of NMC-A was solved to 1.64-Å 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 β-lactamases. These unusual features in class A β-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 β-lactamases, such as cephamycins and carbapenems, need to be accommodated.

Antithrombin III: structural and functional aspects.

Antithrombin III is a plasma glycoprotein responsible for thrombin inhibition in the blood coagulation cascade. The X-ray structure of its cleaved form has been determined and refined to 3.2 A resolution. The overall topology is similar to that of alpha 1-antitrypsin, another member of the serpin (serine protease inhibitor) superfamily. The biological activity of antithrombin III is mediated by a polysaccharide, heparin. The binding site of this effector is described. A possible structural transition from the native to the cleaved structure is discussed.

A Novel Protein Fold and Extreme Domain Swapping in the Dimeric TorD Chaperone from Shewanella massilia

TorD is the cytoplasmic chaperone involved in the maturation of the molybdoenzyme TorA prior to the translocation of the folded protein into the periplasm. The X-ray structure at 2.4 A resolution of the TorD dimer reveals extreme domain swapping between the two subunits. The all-helical architecture of the globular domains within the intertwined molecular dimer shows no similarity with known protein structures. According to sequence similarities, this new fold probably represents the architecture of the chaperones associated with the bacterial DMSO/TMAO reductases and also that of proteins of yet unknown functions. The occurrence of multiple oligomeric forms and the chaperone activity of both monomeric and dimeric TorD raise questions about the possible biological role of domain swapping in this protein.

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.

β-lactamase TEM1 of E. coli Crystal structure determination at 2.5 Å resolution

The crystal structure of β‐lactamase TEM1 from E. coli has been solved to 2.5 Å resolution by X‐ray diffraction methods and refined to a crystallographic R‐factor of 22.7%. The structure was determined by multiple isomorphous replacement using four heavy atom derivatives. The solution from molecular replacement, using a polyalanine model constructed from the Cα coordinates of S. Aureus PC1 enzyme, provided a set of phases used for heavy atom derivatives analysis. The E. coli β‐lactamase TEM1 is made up of two domains whose topology is similar to that of the PC1 enzyme. However, global superposition or the two proteins shows significant differences.

Cardiotoxin VII4 from Naja mossambica mossambica. The refined crystal structure.

The crystal structure of cardiotoxin VII4 from Naja mossambica mossambica was refined to 2.5 A resolution. Fifty ordered solvent sites were localized and included in the refinement. The final R factor is 0.197 (lambda/(2sin theta) less than 5 A; F greater than 3 sigma). The three-dimensional structure is characterized by two beta-sheets. Of particular interest is the two-stranded beta-sheet in the N-terminal region. This shows a large right-handed twist and, though strongly connected to the core of the molecule, and in particular to the C-terminal end, protrudes out of the bulk of the molecule. The segment of four amino acid residues connecting the two strands of this sheet is particularly exposed. It contains an invariant proline residue that has probably an important structural role, and is completely hydrophobic. Two other conserved hydrophobic zones were identified; the largest extends over the second and third loops, on one side only of the molecule. All side-chains of invariant hydrophobic character (except proline residues) belong to one of these three zones. Also discussed are the dimeric assembly and the rather loose packing in the crystal. The three-dimensional structure is compared with that of short and long alpha-neurotoxins. Comparison with two-dimensional nuclear magnetic resonance results on the 68% homologous cardiotoxin CT X IIb shows an excellent overall agreement. A few differences are probably genuine.