How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

6 pags, 4 figs, 1 tabThe expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The highmolecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain - a remarkable 60 Å distant from the DD-transpeptidase active site - discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.Work in the United States was supported by National Institutes of Health Grants AI090818 and AI104987, and work in Spain was supported by Grants BFU2011-25326 (from the Spanish Ministry of Economy and Competitiveness) and S2010/BMD-2457 (from the Autonomous Government of Madrid)

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of L,D-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known L,D-transpeptidases. Mass analyses confirm that Csd6 functions only as an L,D-carboxypeptidase and not as an L,D-transpeptidase. The D-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of L,D-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.1165Ysciescopu

SYNTHESIS, COORDINATION AND REDOX PROPERTIES OF A NOVEL TETRATHIAFULVALENE TETRA(BENZO-15-CROWN-5)ETHER LIGAND

A new tetrathiafulvalene tetra(benzo-15-crown-5)ether ligand (4) has been prepared and polymetallic alkali metal complexes of 4·4Na+ and 4·2K+ stoichiometries isolated. Relatively small anodic perturbations of the redox-active ligand's redox couples were observed in the presence of either alkali metal cation. © 1995

A new carboxylate anion selective cobaltocenium calix[4]arene receptor

A new upper-rim cobaltocenium bridged calix[4]arene receptor L is synthesised and by virtue of its unique topology exhibits remarkable carboxylate anion selectivity

Acid catalysed condensation reactions of 1,3-dihydroxybenzene with new redox-active metallocene aldehydes

New ferrocene and cobaltocenium acetal and aldehyde containing compounds have been prepared. The acid catalysed condensations of both metallocene bis(aldehydes) (5, 6) with 1,3-dihydroxybenzene yielded complex products. The new metallocene cyclic monoaldehydes (7, 8), differed markedly in their reactions with 1,3-dihydroxybenzene with the ferrocene derivative 8 producing [1 + 1] and [1 + 2] bisphenol: aldehyde adducts, whereas the cobaltocenium analogue gave a cyclic diene compound (16). A single crystal X-ray structure of 16 is also described and the electrochemical properties of some of the new metallocene derivatives reported

Crystal structure of N-cyclooctylidene-N-(2,4-dinitrophenyl)-hydrazine

The title compound was prepared from cyclooctenone and 2,4-dinitrophenylhydrazine in ethanol. Recrystallization from diethylether yielded 85% of yellowish prisms

Crystal structure of N-cyclooctylidene-N-(2,4-dinitrophenyl)-hydrazine

The title compound was prepared from cyclooctenone and 2,4-dinitrophenylhydrazine in ethanol. Recrystallization from diethylether yielded 85% of yellowish prisms