Sticholysin II (StnII) is a pore-forming protein (PFP) produced by the sea anemone Stichodactyla helianthus. We found out that StnII exists in a monomeric soluble state but forms tetramers in the presence of a lipidic interface. Both structures have been independently determined at 1.7 A and 18 A resolution, respectively, by using X-ray crystallography and electron microscopy of two-dimensional crystals. Besides, the structure of soluble StnII complexed with phosphocholine, determined at 2.4 A resolution, reveals a phospholipid headgroup binding site, which is located in a region with an unusually high abundance of aromatic residues. Fitting of the atomic model into the electron microscopy density envelope suggests that while the beta sandwich structure of the protein remains intact upon oligomerization, the N-terminal region and a flexible and highly basic loop undergo significant conformational changes. These results provide the structural basis for the membrane recognition step of actinoporins and unexpected insights into the oligomerization step.

The 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 high-molecular 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.

The bacterial thermoalkalophilic lipases that hydrolyze saturated fatty acids at 60 -75°C and pH 8 -10 are grouped as the lipase family I.5. We report here the crystal structure of the lipase from Geobacillus thermocatenulatus, the first structure of a member of the lipase family I.5 showing an open configuration. Unexpectedly, enzyme activation involves large structural rearrangements of around 70 amino acids and the concerted movement of two lids, the ␣6-and ␣7-helices, unmasking the active site. Central in the restructuring process of the lids are both the transfer of bulky hydrophobic residues out of the N-terminal end of the ␣6-helix and the incorporation of short side chain residues to the ␣6 C-terminal end. All these structural changes are stabilized by the Zn 2؉ -binding domain, which is characteristic of this family of lipases. Two detergent molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acids chains. The combination of structural and biochemical studies indicate that the lid opening is not mediated by temperature but triggered by interaction with lipid substrate.

The bactericidal activity of bacteriophages has been used to treat human infections for years as an alternative or a complement to antibiotic therapy. Nowadays, endolysins (phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle) have been used successfully to control antibiotic-resistant pathogenic bacteria in animal models. Their cell wall binding domains target the enzymes to their substrate, and their corresponding catalytic domains are able to cleave bonds in the peptidoglycan network. Recent research has not only revealed the surprising rich structural catalytic diversity of these murein hydrolases but has also yielded insights into their modular organization, their three-dimensional structures, and their mechanism of recognition of bacterial cell wall. These results allow endolysins to be considered as effective antimicrobials with potentially important applications in medicine and biotechnology.

Pneumococcal bacteriophage-encoded lysins are modular choline binding proteins that have been shown to act as enzymatic antimicrobial agents (enzybiotics) against streptococcal infections. Here we present the crystal structures of the free and choline bound states of the Cpl-1 lysin, encoded by the pneumococcal phage Cp-1. While the catalytic module displays an irregular (beta/alpha)(5)beta(3) barrel, the cell wall-anchoring module is formed by six similar choline binding repeats (ChBrs), arranged into two different structural regions: a left-handed superhelical domain configuring two choline binding sites, and a beta sheet domain that contributes in bringing together the whole structure. Crystallographic and site-directed mutagenesis studies allow us to propose a general catalytic mechanism for the whole glycoside hydrolase family 25. Our work provides the first complete structure of a member of the large family of choline binding proteins and reveals that ChBrs are versatile elements able to tune the evolution and specificity of the pneumococcal surface proteins.

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Summary High-level resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is due to expression of penicillin-binding protein 2a (PBP2a), a transpeptidase that catalyzes cell-wall crosslinking in the face of the challenge by β-lactam antibiotics. The activity of this protein is regulated by allostery at a site 60 Å distant from the active site, where crosslinking of cell wall takes place. This review discusses the state of knowledge on this important enzyme of cell-wall biosynthesis in MRSA.

The respiratory pathogen Streptococcus pneumoniae has evolved efficient mechanisms to resist oxidative stress conditions and to displace other bacteria in the nasopharynx. Here we characterize at physiological, functional and structural levels two novel surface-exposed thioredoxin-family lipoproteins, Etrx1 and Etrx2. The impact of both Etrx proteins and their redox partner methionine sulfoxide reductase SpMsrAB2 on pneumococcal pathogenesis was assessed in mouse virulence studies and phagocytosis assays. The results demonstrate that loss of function of either both Etrx proteins or SpMsrAB2 dramatically attenuated pneumococcal virulence in the acute mouse pneumonia model and that Etrx proteins compensate each other. The deficiency of Etrx proteins or SpMsrAB2 further enhanced bacterial uptake by macrophages, and accelerated pneumococcal killing by H2O2 or free methionine sulfoxides (MetSO). Moreover, the absence of both Etrx redox pathways provokes an accumulation of oxidized SpMsrAB2 in vivo. Taken together our results reveal insights into the role of two extracellular electron pathways required for reduction of SpMsrAB2 and surface-exposed MetSO. Identification of this system and its target proteins paves the way for the design of novel antimicrobials.