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Funding: The authors gratefully acknowledge the China Scholarship Council for funding a PhD scholarship for D. W. This work was also funded by the Leverhulme Trust (grant no. RPG-2017-214). I. G. S., W. M. and M. G. were funded by NIH grant R01-GM132595 and Harrison Family Funds. M. P. C. acknowledges the support of a Robert R. Wagner Fellowship at the University of Virginia. R. F. was supported by the Biological and Biotechnological Sciences Research Council (grant no. BB/V014684/1). Serum albumin–Co2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin–Co2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co2+. Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co2+-binding sites (site A), respectively. The presence of additional multiple weak-affinity Co2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co2+-binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co2+. Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co2+ binding to serum albumin. Publisher PDF

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.

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 PDF

The five-membered nitrogen plus heteroatom rings known as azolines or in their oxidized form as azoles are very common in natural products and drugs. The oxidation of thiazoline to thiazole in the cyanobactin class of natural products is one of the several important transformations that are known to alter the biological properties of the compound. The ordering of the various chemical reactions that occur during cyanobactin biosynthesis is not fully understood. The structure of the flavin-dependent enzyme responsible for the oxidation of multiple thiazolines reveals it contains an additional domain that in other enzymes recognizes linear peptides. We characterize the activity of the enzyme on two substrates: one with a peptide leader and one without. Kinetics and biophysics reveal that the leader on the substrate is not recognized by the enzyme. The enzyme is faster on either substrate than the macrocyclase or protease in vitro. The enzyme has a preferred order of oxidation of multiple thiazolines in the same linear peptide. Postprint

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 work was funded by the Raymond and Beverley Sackler Institute for Biological, Physical, and Engineering sciences [to L.R.]; the National Institute of Health [Grant nos. GM118528 and CA209992 to M. H. and L. R.]; the Medical Research Council [Grant no. MR/K001485 to U.S.L. and J. M. E.]; a Leverhulme Trust Visiting Professorship [to L. R.]; and Royal Society of Edinburgh [Caledonian Scholarship to O.K.M.]. We present a novel method to fluorescently label proteins, post-translationally, within live Saccharomycescerevisiae. The premise underlying this work is that fluorescent protein (FP) tags are less disruptive to normal processing and function when they are attached post-translationally, because target proteins are allowed to fold properly and reach their final subcellular location before being labeled. We accomplish this post-translational labeling by expressing the target protein fused to a short peptide tag (SpyTag), which is then covalently labeled in situ by controlled expression of an open isopeptide domain (SpyoIPD, a more stable derivative of the SpyCatcher protein) fused to an FP. The formation of a covalent bond between SpyTag and SpyoIPD attaches the FP to the target protein. We demonstrate the general applicability of this strategy by labeling several yeast proteins. Importantly, we show that labeling the membrane protein Pma1 in this manner avoids the mislocalization and growth impairment that occur when Pma1 is genetically fused to an FP. We also demonstrate that this strategy enables a novel approach to spatiotemporal tracking in single cells and we develop a Bayesian analysis to determine the protein’s turnover time from such data. Postprint

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.

Staphylococcus aureus can adhere to and invade endothelial cells by binding to the human protein fibronectin (Fn). FnBPA and FnBPB, cell wall-attached proteins from S. aureus , have multiple, intrinsically disordered, high-affinity binding repeats (FnBRs) for Fn. Here, 30 years after the first report of S. aureus /Fn interactions, we present four crystal structures that together comprise the structures of two complete FnBRs, each in complex with four of the N-terminal modules of Fn. Each ≈40-residue FnBR forms antiparallel strands along the triple-stranded β-sheets of four sequential F1 modules ( 2–5 F1) with each FnBR/ 2–5 F1 interface burying a total surface area of ≈4,300 Å 2 . The structures reveal the roles of residues conserved between S. aureus and Streptococcus pyogenes FnBRs and show that there are few linker residues between FnBRs. The ability to form large intermolecular interfaces with relatively few residues has been proposed to be a feature of disordered proteins, and S. aureus /Fn interactions provide an unusual illustration of this efficiency.

Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells1, 2. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats3 in an unfolded4, 5 region of the protein. The bacterium-binding site in the amino-terminal domain (1–5F1) of Fn contains five sequential Fn type 1 (F1) modules. Here we show the structure of a streptococcal (S. dysgalactiae) FnBP peptide (B3)6, 7 in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel -strands on sequential F1 modules—the first example of a tandem -zipper. Sequence analyses of larger regions of FnBPs from S. pyogenes and S. aureus reveal a repeating pattern of F1-binding motifs that match the pattern of F1 modules in 1–5F1 of Fn. In the process of Fn-mediated invasion of host cells, therefore, the bacterial proteins seem to exploit the modular structure of Fn by forming extended tandem -zippers. This work is a vital step forward in explaining the full mechanism of the integrin-dependent2, 8 FnBP-mediated invasion of host cells.

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.

The SSPF was supported by grants from The Scottish Funding Council (references SSPF and SULSA), The Biotechnology and Biological Sciences Research Council (reference BB/S/B14450), European Union under framework 7 (reference Aeropath). The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to high throughput structure determination. The effort was successful in that over 40 structures were determined. These structures and the methods harnessed to obtain them are reported here. This report reflects on the value of automation but also on the continued requirement for a high degree of scientific and technical expertise. The efficiency of the process poses challenges to the current paradigm of structural analysis and publication. In the 5 year period we published ten peer-reviewed papers reporting structural data arising from the pipeline. Nevertheless, the number of structures solved exceeded our ability to analyse and publish each new finding. By reporting the experimental details and depositing the structures we hope to maximize the impact of the project by allowing others to follow up the relevant biology. Publisher PDF

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.

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

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

Integrin-dependent cell invasion of some pathogenic bacteria is mediated by surface proteins targeting the extracellular matrix protein fibronectin (FN). Although the structural basis for bacterial FN recognition is well understood, it has been unclear why proteins such as streptococcal SfbI contain several FN-binding sites. We used microcalorimetry to reveal cooperative binding of FN fragments to arrays of binding sites in SfbI. In combination with thermodynamic analyses, functional cell-based assays show that SfbI induces conformational changes in the N-terminal 100-kDa region of FN (FN100kDa), most likely by competition with intramolecular interactions defining an inactive state of FN100kDa. This study provides insights into how long range conformational changes resulting in FN activation may be triggered by bacterial pathogens.

Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate invasion of human endothelial and epithelial cells in a process likely to aid the persistence and/or dissemination of infection. In addition to binding sites for the N-terminal domain (NTD) of fibronectin (Fn), a number of streptococcal FnBPs also contain an upstream region (UR) that is closely associated with an NTD-binding region; UR binds to the adjacent gelatin-binding domain (GBD) of Fn. Previously, UR was shown to be required for efficient streptococcal invasion of epithelial cells. Here we show, using a Streptococcus zooepidemicus FnBP, that the UR-binding site in GBD resides largely in the (8)F1(9)F1 module pair. We also show that UR inhibits binding of a peptide from the α1 chain of type I collagen to (8)F1(9)F1 and that UR binding to (8)F1 is likely to occur through anti-parallel β-zipper formation. Thus, we propose that streptococcal proteins that contain adjacent NTD- and GBD-binding sites form a highly unusual extended tandem β-zipper that spans the two domains and mediates high affinity binding to Fn through a large intermolecular interface. The proximity of the UR- and NTD-binding sequences in streptococcal FnBPs is consistent with a non-linear arrangement of modules in the tertiary structure of the GBD of Fn.

Xeroderma pigmentosum factor D (XPD) is a 5'-3' superfamily 2 helicase and the founding member of a family of DNA helicases with iron-sulphur cluster domains. As a component of transcription factor II H (TFIIH), XPD is involved in DNA unwinding during nucleotide excision repair (NER). Archaeal XPD is closely related in sequence to the eukaryal enzyme and the crystal structure of the archaeal enzyme has provided a molecular understanding of mutations causing xeroderma pigmentosum and trichothiodystrophy in humans. Consistent with a role in NER, we show that archaeal XPD can initiate unwinding from a DNA bubble structure, differentiating it from the related helicases FancJ and DinG. XPD was not stalled by substrates containing extrahelical fluorescein adducts, abasic sites nor a cyclobutane pyrimidine dimer, regardless of whether these modifications were placed on either the displaced or translocated strands. This suggests that DNA lesions repaired by NER may not present a barrier to XPD translocation in vivo, in contrast to some predictions. Preferential binding of a fluorescein-adducted oligonucleotide was observed, and XPD helicase activity was readily inhibited by both single- and double-stranded DNA binding proteins. These observations have several implications for the current understanding of the NER pathway. Publisher PDF

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.

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.

Host invasion by a number of pathogenic bacteria such as staphylococci and streptococci involves binding to fibronectin, a ubiquitous extracellular matrix protein. On the bacterial side, host extracellular matrix adherence is mediated by MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) which, in some cases, have been identified to be important virulence factors. In this study we used nuclear magnetic resonance spectroscopy to characterize the interaction of B3, a synthetic peptide derived from an adhesin of Streptococcus dysgalactiae, with the N-terminal module pair 1F12F1 of human fibronectin. 1F12F1 chemical shift changes occurring on formation of the 1F12F1/B3 complex indicate that both modules bind to the peptide and that a similar region of each module is involved. A similar surface of the 4F15F1 module pair had previously been identified as the binding site for a fibronectin-binding peptide from Staphylococcus aureus.

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence., eLife digest Rift Valley fever phlebovirus (RVFV) is a virus of humans and livestock, transmitted by mosquitos and contact with infected animals. Infection can cause severe disease, including hemorrhagic fever, and may lead to death. Historically, the virus was only found in central Africa but it has spread for instance to the Arabian Peninsula. There is a risk that the virus may appear in temperate regions including Europe because global warming is allowing the mosquitos that carry the virus to extend their geographic range. There are no vaccines or treatments available for use in humans so if there is a serious outbreak of the virus it could become an epidemic and cause great economic losses and severe human disease. RVFV relies on a protein called NSs to cause disease. In cells of infected animals and humans NSs forms filaments inside the nucleus, the control center of the cell, and disarms the immune system. However, it is not known precisely how NSs works. To address this question, Barski, Brennan et al. used a technique called X-ray crystallography to study the atomic three-dimensional structure of NSs. This revealed that the center of the protein contains a core domain that causes the filaments to form. Further experiments identified how the NSs core comes together to build the filaments inside the cell nucleus. These findings represent an important step towards understanding how the NSs protein helps RVFV to cause disease in humans and livestock. In the future, this work may aid the development of much needed drugs and vaccines against RVFV.