A new structural class of bacterial thioester domains reveals a slipknot topology

This work was supported by the MRC, UK grant MR/K001485 for MJB, USL; the BBSRC, UK grant BB/J00453 and the John Innes Foundation for MJB; The Royal Society of Edinburgh and the Carnegie Trust for OKM.An increasing number of surface‐associated proteins identified in Gram‐positive bacteria are characterized by intramolecular cross‐links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of Class I structurally characterized. Here, we present crystal structures of three Class II TEDs from Bacillus anthracis, vancomycin‐resistant Staphylococcus aureus, and vancomycin‐resistant Enterococcus faecium. These proteins are structurally distinct from Class I TEDs due to a β‐sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full‐length sortase‐anchored protein structure (BaTIE). This provides insight into the three‐dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram‐positive bacteria.Publisher PDFPeer reviewe

Insights into the mechanism of the cyanobactin heterocyclase enzyme

The work is supported by the European Research Council NCB-TNT (339367), Biotechnology and Biological Sciences Research Council (BB/K015508/1 and BB/M001679/1).Cyanobactin heterocyclases share the same catalytic domain (YcaO) as heterocyclases/cyclodehydratases from other ribosomal peptide (RiPPs) biosynthetic pathways. These enzymes process multiple residues (Cys/Thr/Ser) within the same substrate. The processing of cysteine residues proceeds with a known order. We show the order of reaction for threonines is different and depends in part on a leader peptide within the substrate. In contrast to other YcaO domains, which have been reported to exclusively break down ATP into ADP and inorganic phosphate, cyanobactin heterocyclases have been observed to produce AMP and inorganic pyrophosphate during catalysis. We dissect the nucleotide profiles associated with heterocyclization and propose a unifying mechanism, where the γ-phosphate of ATP is transferred in a kinase mechanism to the substrate to yield a phosphorylated intermediate common to all YcaO domains. In cyanobactin heterocyclases, this phosphorylated intermediate, in a proportion of turnovers, reacts with ADP to yield AMP and pyrophosphate.Publisher PDFPeer reviewe

Plasma free fatty acid levels influence Zn2+-dependent histidine-rich glycoprotein-heparin interactions via an allosteric switch on serum albumin

Background: Histidine-rich glycoprotein (HRG) regulates coagulation, through its ability to bind and neutralize heparins. HRG associates with zinc ions (Zn2+) to stimulate HRG-heparin complex formation. Under normal conditions the majority of plasma Zn2+ associates with human serum albumin (HSA). However, free fatty acids (FFA) allosterically disrupt Zn2+ binding to HSA. Thus high levels of circulatory FFA, as are associated with diabetes, obesity and cancer, may increase the proportion of plasma Zn2+ associated with HRG contributing to an increased risk of thrombotic disease. Objectives: The aims were to characterize Zn2+ binding by HRG, examine the influence FFA have on Zn2+ binding by HSA and establish whether FFA-mediated displacement of Zn2+ from HSA may influence HRG-heparin complex formation. Methods: Zn2+ binding to HRG and to HSA in the presence of different FFA (myristate) concentrations were examined by isothermal titration calorimetry (ITC) and the formation of HRG-heparin complexes in the presence of different Zn2+ concentrations by both ITC and an ELISA-based assay. Results and conclusions: We reveal that HRG possesses 10 Zn2+ sites (K´=1.63 x 105) and that cumulative binding of FFA to HSA perturbed its ability to bind Zn2+. Also Zn2+ binding was shown to increase the affinity by which HRG interacts with unfractionated heparins but has no effect upon its interaction with low molecular weight heparin (ca. 6850 kDa). Speciation modeling of plasma Zn2+ based upon the data obtained suggests that FFA-mediated displacement of Zn2+ from serum albumin would be likely to contribute to the development of thrombotic complications in individuals with high plasma FFA levels.Publisher PDFPeer reviewe

Fibronectin-binding proteins of Gram-positive cocci

Cell wall-attached fibronectin-binding proteins are important multifunctional virulence factors of Staphylococcus aureus and Streptococcus pyogenes. This review describes recent advances in the understanding of the function of these proteins on a molecular level and of their role in infections. (c) 2006 Elsevier SAS. All rights reserved.</p

The molecular basis of fibronectin-mediated bacterial adherence to host cells

Many pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to components of the extracellular matrix (ECM) of the host. These bacterial MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are thought to play a critical role in infection. One group of MSCRAMMs, produced by staphylococci and streptococci, targets fibronectin (Fn, a glycoprotein found in the ECM and body fluids of vertebrates) using repeats in the C-terminal region of the bacterial protein. These bacterial Fn-binding proteins (FnBPs) mediate adhesion to host tissue and bacterial uptake into non-phagocytic host cells. Recent studies on interactions between the host and bacterial proteins at the residue-specific level and on the mechanism of host cell invasion are providing a much clearer picture of these processes.</p

The molecular basis of fibronectin-mediated bacterial adherence to host cells

Many pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to components of the extracellular matrix (ECM) of the host. These bacterial MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) are thought to play a critical role in infection. One group of MSCRAMMs, produced by staphylococci and streptococci, targets fibronectin (Fn, a glycoprotein found in the ECM and body fluids of vertebrates) using repeats in the C-terminal region of the bacterial protein. These bacterial Fn-binding proteins (FnBPs) mediate adhesion to host tissue and bacterial uptake into non-phagocytic host cells. Recent studies on interactions between the host and bacterial proteins at the residue-specific level and on the mechanism of host cell invasion are providing a much clearer picture of these processes.</p

Fibronectin-binding proteins of Gram-positive cocci

Cell wall-attached fibronectin-binding proteins are important multifunctional virulence factors of Staphylococcus aureus and Streptococcus pyogenes. This review describes recent advances in the understanding of the function of these proteins on a molecular level and of their role in infections. (c) 2006 Elsevier SAS. All rights reserved.</p