Papers by Ulrich Schwarz-Linek
Schwarz-Linek, U. et al. Pathogenic bacteria attach to human fibronectin through a tandem -zipper. Nature 423, 177-181
Nature
Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibr... more Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats in an unfolded 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) in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel beta-strands on sequential F1 modules-the first example of a tandem beta-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 m...
eLife, 2015
To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues.... more To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a 'chemical harpoon'. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportun...
Journal of Thrombosis and Haemostasis, 2014
To cite this article: Kassaar O, Schwarz-Linek U, Blindauer CA, Stewart AJ. Plasma free fatty aci... more To cite this article: Kassaar O, Schwarz-Linek U, Blindauer CA, Stewart AJ. Plasma free fatty acid levels influence Zn 2+ -dependent histidine-rich glycoprotein-heparin interactions via an allosteric switch on serum albumin. J Thromb Haemost 2015; 13: 101-10.
ChemInform Abstract: Enzymatic Baeyer-Villiger Oxidation with Cyclohexanone Monooxygenase: A New System of Cofactor Regeneration
ChemInform, 2000
ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Angewandte Chemie, 2014
Heterocycle-containing cyclic peptides are promising scaffolds for the pharmaceutical industry bu... more 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.
Tetrahedron: Asymmetry, 1997
A two-enzyme system consisting of a cyciohexanone mono-oxygenase from Acinetobacter NCIMB 9871 an... more A two-enzyme system consisting of a cyciohexanone 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 aeration by means of a thin-walled silicon tube.
Synthesis of Natural Product Precursors by Baeyer-Villiger Oxidation with Cyclohexanone Monooxygenase from Acinetobacter
Synthesis, 2001
The Baeyer-Villiger oxidation of the 2-substituted ketones 1 and 3 with the coupled system cycloh... more The Baeyer-Villiger oxidation of the 2-substituted ketones 1 and 3 with the coupled system cyclohexanone monooxygenase from Acinetobacter NCIMB 9871 / formate dehydrogenase from Pseudomonas sp. 101 provides the lactones (R)-2 and (R)-4 with high enantiomeric excess which are ...
Proceedings of the National Academy of Sciences, 2008
FnBPB, cell wall-attached proteins from S. aureus, have multiple, intrinsically disordered, high-... more 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 Ϸ40-residue FnBR forms antiparallel strands along the triple-stranded -sheets of four sequential F1 modules ( 2-5 F1) with each FnBR/ 2-5 F1 interface burying a total surface area of Ϸ4,300 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.
Yet more intramolecular cross-links in Gram-positive surface proteins
Proceedings of the National Academy of Sciences, 2014
Proceedings of the National Academy of Sciences, 2012
Protein interactions with peptides generally have low thermodynamic and mechanical stability. Str... more 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 (Spy-Catcher) 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.
Nucleic Acids Research, 2010
Xeroderma pigmentosum factor D (XPD) is a 5 0 -3 0 superfamily 2 helicase and the founding member... more Xeroderma pigmentosum factor D (XPD) is a 5 0 -3 0 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 doublestranded DNA binding proteins. These observations have several implications for the current understanding of the NER pathway.
Pathogenic bacteria attach to human fibronectin through a tandem β-zipper
Nature, 2003
Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibr... more Staphylococcus aureus and Streptococcus pyogenes, two important human pathogens, target host fibronectin (Fn) in their adhesion to and invasion of host cells. Fibronectin-binding proteins (FnBPs), anchored in the bacterial cell wall, have multiple Fn-binding repeats in an unfolded 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) in complex with the module pair 1F12F1. This identifies 1F1- and 2F1-binding motifs in B3 that form additional antiparallel beta-strands on sequential F1 modules-the first example of a tandem beta-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 beta-zippers. This work is a vital step forward in explaining the full mechanism of the integrin-dependent FnBP-mediated invasion of host cells.
Molecular Microbiology, 2004
Many pathogenic Gram-positive bacteria produce cell wall-anchored proteins that bind to component... more 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 nonphagocytic 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.
Fibronectin-binding proteins of Gram-positive cocci
Microbes and Infection, 2006
Cell wall-attached fibronectin-binding proteins are important multifunctional virulence factors o... more 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.
The Scottish Structural Proteomics Facility: targets, methods and outputs
Journal of Structural and Functional Genomics, 2010
The Scottish Structural Proteomics Facility was funded to develop a laboratory scale approach to ... more 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.
The Solution and Crystal Structures of a Module Pair from the Staphylococcus aureus-Binding Site of Human Fibronectin—A Tale with a Twist
Journal of Molecular Biology, 2007
An important goal of structural studies of modular proteins is to determine the inter-module orie... more 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.
The Streptococcal Binding Site in the Gelatin-binding Domain of Fibronectin Is Consistent with a Non-linear Arrangement of Modules
Journal of Biological Chemistry, 2010
Fibronectin-binding proteins (FnBPs) of Staphylococcus aureus and Streptococcus pyogenes mediate ... more 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.
BBK32, a Fibronectin Binding MSCRAMM from Borrelia burgdorferi, Contains a Disordered Region That Undergoes a Conformational Change on Ligand Binding
Journal of Biological Chemistry, 2004
BBK32 is a fibronectin-binding lipoprotein on Borrelia burgdorferi, the causative agent of Lyme d... more 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 alpha-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 beta-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 beta-zipper" previously demonstrated for the fibronectin binding of streptococcal adhesins.
High Affinity Streptococcal Binding to Human Fibronectin Requires Specific Recognition of Sequential F1 Modules
Journal of Biological Chemistry, 2004
Fibronectin (Fn) binding by the Streptococcus pyogenes protein SfbI has been shown to trigger int... more 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.
The Tandem beta-Zipper Model Defines High Affinity Fibronectin-binding Repeats within Staphylococcus aureus FnBPA
Journal of Biological Chemistry, 2007
Binding of the fibronectin-binding protein FnBPA from Staphylococcus aureus to the human protein ... more 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.
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Papers by Ulrich Schwarz-Linek