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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.

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.

This paper was funded by: Funded Access, Leverhulme Trust. Grant Number: RPG-2012-504, TSB. Grant Number: 131181, ERC. Grant Number: 339367, and BBSRC. Grant Number: BB/K015508/1 Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry but their chemical synthesis is very challenging. A new universal method has been devised to prepare these compounds by using a set of engineered marine-derived enzymes and substrates obtained from a family of ribosomally produced and post-translationally modified peptides called the cyanobactins. The substrate precursor peptide is engineered to have a non-native protease cleavage site that can be rapidly cleaved. The other enzymes used are heterocyclases that convert Cys or Cys/Ser/Thr into their corresponding azolines. A macrocycle is formed using a macrocyclase enzyme, followed by oxidation of the azolines to azoles with a specific oxidase. The work is exemplified by the production of 17 macrocycles containing 6-9 residues representing 11 out of the 20 canonical amino acids. Publisher PDF

Counting backwards: The cyanobactin class of heterocyclases, exemplified by TruD, possess an almost unique combination of processivity, specificity, chemical versatility, and promiscuity. TruD is shown by biochemical assay to be an adenylase, and processes cysteines in a defined order. The entire substrate leader can be removed and TruD will process a single specific cysteine residue; however the role of leader is to permit processivity through a balance of recognition. ATP/AMP=adenosine tri/monophosphate; PPi=pyrophosphate.

Collagen and fibronectin (FN) are two abundant and essential components of the vertebrate extracellular matrix; they interact directly with cellular receptors and affect cell adhesion and migration. Past studies identified a FN fragment comprising six modules, (6)FnI(1-2)FnII(7-9)FnI, and termed the gelatin binding domain (GBD) as responsible for collagen interaction. Recently, we showed that the GBD binds tightly to a specific site within type I collagen and determined the structure of domains (8-9)FnI in complex with a peptide from that site. Here, we present the crystallographic structure of domains (6)FnI(1-2)FnII(7)FnI, which form a compact, globular unit through interdomain interactions. Analysis of NMR titrations with single-stranded collagen peptides reveals a dominant collagen interaction surface on domains (2)FnII and (7)FnI; a similar surface appears involved in interactions with triple-helical peptides. Models of the complete GBD, based on the new structure and the (8-9)FnI·collagen complex show a continuous putative collagen binding surface. We explore the implications of this model using long collagen peptides and discuss our findings in the context of FN interactions with collagen fibrils.

[**]We thank Dr D. Uhrin (Edinburgh) and Dr T. Lebl for NMR advice, Dr C. Botting for MS, M. Taylor for help with thermal shiftassays and Dr S. Talay (Braunschweig) for her kind gift of S. pyogenes DNA. MB and RB wish to thank EaStCHEM for support andfor access to the Research Computing Facility, and Dr H. Fruchtl for technical assistance. This work was funded in part by a Value inPeople award of the Wellcome Trust.

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.

Staphylococcus aureus possesses cell-wall attached proteins that bind the human protein fibronectin (Fn). An intermodule interface between the 4F1 and 5F1 modules in the N-terminal domain of Fn is maintained on bacterial peptide binding but there is a small change in the intermodule orientation and alignment of β-strands that are predicted to bind the peptide. The module pair is elongated, as in the unbound state. Combined with evidence that residues in both 4F1 and 5F1 are directly involved in peptide binding, this observation supports the hypothesis that, when bound to intact Fn, the bacterial protein adopts an unusual, highly extended conformation.

BBK32 is a fibronectin-binding protein from the Lyme disease-causing spirochete Borrelia burgdorferi. In this study, we show that BBK32 shares sequence similarity with fibronectin module-binding motifs previously identified in proteins from Streptococcus pyogenes and Staphylococcus aureus. Nuclear magnetic resonance spectroscopy and isothermal titration calorimetry are used to confirm the binding sites of BBK32 peptides within the N-terminal domain of fibronectin and to measure the affinities of the interactions. Comparison of chemical shift perturbations in fibronectin F1 modules on binding of peptides from BBK32, FnBPA from S. aureus, and SfbI from S. pyogenes provides further evidence for a shared mechanism of binding. Despite the different locations of the bacterial attachment sites in BBK32 compared with SfbI from S. pyogenes and FnBPA from S. aureus, an antiparallel orientation is observed for binding of the N-terminal domain of fibronectin to each of the pathogens. Thus, these phylogenetically and morphologically distinct bacterial pathogens have similar mechanisms for binding to human fibronectin.

BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme disease. Analysis using secondary structure prediction programs suggested that BBK32 is composed of two domains, an N-terminal segment lacking well defined secondary structure and a C-terminal segment composed largely of α-helices. Analysis of purified recombinant forms of the two domains by circular dichroism spectroscopy, gel permeation chromatography, and intrinsic viscosity determination were consistent with an N-terminal-extended, unstructured segment and a C-terminal globular domain in BBK32. Solid phase binding experiments suggest that the unstructured N-terminal domain binds fibronectin. Analysis of changes in circular dichroism spectra of the N-terminal segment of BBK32 upon binding of the N-terminal domain of fibronectin revealed an increase in β-sheet content in the complex. Hence, BBK32, which belongs to a different family of proteins and shows no overall sequence similarity with the fibronectin binding MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) of Gram-positive bacteria, binds fibronectin by a mechanism that is reminiscent of the “tandem β-zipper” previously demonstrated for the fibronectin binding of streptococcal adhesins (Schwarz-Linek, U., Werner, J. M., Pickford, A. R., Gurusiddappa, S., Kim, J. H., Pilka, E. S., Briggs, J. A., Gough, T. S., Hook, M., Campbell, I. D., and Potts, J. R. (2003) Nature 423, 177–181).

Summary 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.

A two-enzyme system consisting of a cyclohexanone mono-oxygenase from Acinetobacter NCIMB 9871 and a protein-engineered formate dehydrogenase for the regeneration of the cofactor NADPH was used for the synthesis of chiral e-lactones. 4-Methylcyclohexanone was used as the model substrate yielding ( S )-(−)-5-methyl-oxepane-2-one with high chemical and enantiomeric purity. Syntheses were carried out in a repetitive-batch reactor with integrated bubble-free aerationby means of a thin-walled silicon tube.

The surface of Gram-positive bacteria comprises a single membrane and, typically, a thick layer of cross-linked peptidoglycan that imparts strength and rigidity. Anchored to this cell wall are protein assemblies, such as pili, and surface proteins, such as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that act as surface adhesins. These proteins are critical not only for bacterial binding to host surfaces in the early stages of infection but also for biofilm formation and immune evasion. In a series of studies published since 2007, these cell surface proteins and protein assemblies have been shown to contain isopeptide and thioester bonds, highly unusual intramolecular covalent linkages between amino acid side chains. These cross-links have either a structure-stabilizing role or may be directly involved in adhesion. Using an elegant combination of structural biology and mass spectrometry, Kwon et al. (1) reveal in PNAS an ester bond as yet another covalent linkage in a putative MSCRAMM from the Gram-positive pathogen Clostridium perfringens . Remarkably, this ester bond, joining the side chains of a Thr and Gln residue, is equivalent to an unresolved acyl-enzyme intermediate, formed on an autocatalytic pathway that resembles the mechanism of serine proteases. Despite fundamental differences in the way they are encoded in bacterial genomes, following delivery/assembly at the cell surface, pili and many MSCRAMMs share important similarities. In such proteins, a series of repetitive domains (commonly referred to as stalk regions) are responsible for the length of the structure and allow an N-terminal region to be positioned away from the cell surface. At the C terminus, both pili and MSCRAMMs are anchored to the cell wall by sortases (2). Although many of the building blocks of Gram-positive surface proteins and pili bear some resemblance to their Gram-negative … [↵][1]1To whom correspondence should be addressed. E-mail: Mark.Banfield{at}jic.ac.uk. [1]: #xref-corresp-1-1

Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein fibronectin has previously been implicated in the development of infective endocarditis, specifically in the processes of platelet activation and invasion of the endothelium. We recently proposed a model for binding of fibronectin to FnBPA in which the bacterial protein contains 11 potential binding sites (FnBPA-1 to FnBPA-11), each composed of motifs that bind to consecutive fibronectin type 1 modules in the N-terminal domain of fibronectin. Here we show that six of the 11 sites bind with dissociation constants in the nanomolar range; other sites bind more weakly. The high affinity binding sites include FnBPA-1, the sequence of which had previously been thought to be encompassed by the fibrinogen-binding A domain of FnBPA. Both the number and sequence conservation of the type-1 module binding motifs appears to be important for high affinity binding. The in vivo relevance of the in vitro binding studies is confirmed by the presence of antibodies in patients with S. aureus infections that specifically recognize complexes of these six high affinity repeats with fibronectin.

An important goal of structural studies of modular proteins is to determine the inter-module orientation, which often influences biological function. The N-terminal domain of human fibronectin (Fn) is composed of a string of five type 1 modules (F1). Despite their small size, to date F1 modules have proved intractable to X-ray structure solution, although there are several NMR structures available. Here, we present the first structures (two X-ray models and an NMR-derived model) of the (2)F1(3)F1 module pair, which forms part of the binding site for Fn-binding proteins from pathogenic bacteria. The crystallographic structure determination was aided by the novel technique of UV radiation damage-induced phasing. The individual module structures are very similar in all three models. In the NMR structure and one of the X-ray structures, a similar but smaller interdomain interface than that observed previously for (4)F1(5)F1 is seen. The other X-ray structure has a different interdomain orientation. This work underlines the benefits of combining X-ray and NMR data in the studies of multi-domain proteins.

Fibronectin (Fn) binding by the Streptococcus pyogenes protein SfbI has been shown to trigger integrin-dependent internalization of this pathogen by human epithelial and endothelial cells. Here, using nuclear magnetic resonance spectroscopy and isothermal titration calorimetry in a dissection approach, the basis for the specificity and high affinity of the interaction between the N-terminal domain of Fn and SfbI is revealed. Each of the five Fn type 1 modules is directly involved in the interaction and is recognized by short consecutive motifs within the repeat region of SfbI. Crucially, these motifs must be combined in the correct order to form a high affinity ligand for the N-terminal domain of Fn.

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.

Human fibronectin (Fn) is a large multidomain protein found in the extracellular matrix and plasma. It is involved in many cellular processes, including cell adhesion and migration during embryogenesis and wound healing. The ability to bind Fn is a characteristic that has been demonstrated for a number of pathogens. For Staphylococcus aureus and Streptococcus pyogenes in particular, Fn-binding bacterial proteins (FnBPs) have been shown to mediate not only bacterial adhesion to host cells but also the uptake of bacteria by the cells. FnBPs interact with the amino-terminal region of Fn, where five type I ((1-5)F1) Fn modules are located. Although the structures of two F1 module pairs have been determined by NMR, no X-ray structures have been reported. To explore the conformational interactions between modules and the binding properties of FnBPs, the (2)F1(3)F1 module pair was crystallized using the vapour-diffusion method at 298 K. 12 X-ray diffraction data sets have been collected: six on an in-house rotating anode (three native, one Pt derivative and two peptide-bound) and six at synchrotron-radiation sources (two native and four derivative). Following analysis of these data, some of which have very high multiplicity (up to 50), probable space-group assignments were made (P42(1)2, P4(1)2(1)2 or P4(3)2(1)2) and the possibly twinned nature of the crystals was investigated using six different tests. The results presented here suggest that the crystals are not twinned.