Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family.

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.

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.

The molecular puzzle of two-component signaling cascades.

Two-component systems constitute prevalent signaling pathways in bacteria and mediate a large variety of adaptative cellular responses. Signaling proceeds through His-Asp phosphorelay cascades that involve two central partners, the histidine protein kinase and the response regulator protein. Structural studies have provided insights into some design principles and activation mechanisms of these multi-domain proteins implicated in the control of virulence gene expression in several pathogens.

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.

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.

Two-wavelength MAD phasing: in search of the optimal choice of wavelengths.

The multiwavelength anomalous dispersion (MAD) method is increasingly being used to determine protein crystal structures. In theory, data collection at two wavelengths is sufficient for the determination of MAD phases, but three or even more wavelengths are used most often. In this paper, the results of the phasing procedure using only two wavelengths for proteins containing different types of anomalous scatterers are analyzed. In these cases, it is shown that this approach leads to interpretable maps, similar in quality to those obtained with data collected at three wavelengths, provided that the wavelengths are chosen so as to give a large contrast in the real part of the anomalous scattering factor f. The consequences for a rational MAD data-collection strategy are discussed.

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.

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.

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.