Secreted proteases control autolysin-mediated biofilm growth of Staphylococcus aureus.

Staphylococcus epidermidis, a commensal of humans, secretes Esp protease to prevent Staphylococcus aureus biofilm formation and colonization. Blocking S. aureus colonization may reduce the incidence of invasive infectious diseases; however, the mechanism whereby Esp disrupts biofilms is unknown. We show here that Esp cleaves autolysin (Atl)-derived murein hydrolases and prevents staphylococcal release of DNA, which serves as extracellular matrix in biofilms. The three-dimensional structure of Esp was revealed by x-ray crystallography and shown to be highly similar to that of S. aureus V8 (SspA). Both atl and sspA are necessary for biofilm formation, and purified SspA cleaves Atl-derived murein hydrolases. Thus, S. aureus biofilms are formed via the controlled secretion and proteolysis of autolysin, and this developmental program appears to be perturbed by the Esp protease of S. epidermidis.

Structure of Streptococcus agalactiae tip pilin GBS104: a model for GBS pili assembly and host interactions.

The crystal structure of a 75 kDa central fragment of GBS104, a tip pilin from the 2063V/R strain of Streptococcus agalactiae (group B streptococcus; GBS), is reported. In addition, a homology model of the remaining two domains of GBS104 was built and a model of full-length GBS104 was generated by combining the homology model (the N1 and N4 domains) and the crystal structure of the 75 kDa fragment (the N2 and N3 domains). This rod-shaped GBS104 model is constructed of three IgG-like domains (the N1, N2 and N4 domains) and one vWFA-like domain (the N3 domain). The N1 and N2 domains of GBS104 are assembled with distinct and remote segments contributed by the N- and C-termini. The metal-binding site in the N3 domain of GBS104 is in the closed/low-affinity conformation. Interestingly, this domain hosts two long arms that project away from the metal-binding site. Using site-directed mutagenesis, two cysteine residues that lock the N3 domain of GBS104 into the open/high-affinity conformation were introduced. Both wild-type and disulfide-locked recombinant proteins were tested for binding to extracellular matrix proteins such as collagen, fibronectin, fibrinogen and laminin, and an increase in fibronectin binding affinity was identified for the disulfide-locked N3 domain, suggesting that induced conformational changes may play a possible role in receptor binding.

Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp.

Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P2(1), with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, ß = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Šresolution on a rotating-anode home-source facility.

Structural and biochemical characterization of Staphylococcus aureus clumping factor B/ligand interactions.

Clumping factor B (ClfB) from Staphylococcus aureus is a bifunctional protein that binds to human cytokeratin 10 (K10) and fibrinogen (Fg). ClfB has been implicated in S. aureus colonization of nasal epithelium and is therefore a key virulence factor. People colonized with S. aureus are at an increased risk for invasive staphylococcal disease. In this study, we have determined the crystal structures of the ligand-binding region of ClfB in an apo-form and in complex with human K10 and Fg α-chain-derived peptides, respectively. We have determined the structures of MSCRAMM binding to two ligands with different sequences in the same site showing the versatile nature of the ligand recognition mode of microbial surface components recognizing adhesive matrix molecules. Both ligands bind ClfB by parallel ß-sheet complementation as observed for the clumping factor A·Î³-chain peptide complex. The ß-sheet complementation is shorter in the ClfB·Fg α-chain peptide complex. The structures show that several residues in ClfB are important for binding to both ligands, whereas others only make contact with one of the ligands. A common motif GSSGXG found in both ligands is part of the ClfB-binding site. This motif is found in many human proteins thus raising the possibility that ClfB recognizes additional ligands.

Two autonomous structural modules in the fimbrial shaft adhesin FimA mediate Actinomyces interactions with streptococci and host cells during oral biofilm development.

By combining X-ray crystallography and modelling, we describe here the atomic structure of distinct adhesive moieties of FimA, the shaft fimbrillin of Actinomyces type 2 fimbriae, which uniquely mediates the receptor-dependent intercellular interactions between Actinomyces and oral streptococci as well as host cells during the development of oral biofilms. The FimA adhesin is built with three IgG-like domains, each of which harbours an intramolecular isopeptide bond, previously described in several Gram-positive pilins. Genetic and biochemical studies demonstrate that although these isopeptide bonds are dispensable for fimbrial assembly, cell-cell interactions and biofilm formation, they contribute significantly to the proteolytic stability of FimA. Remarkably, FimA harbours two autonomous adhesive modules, which structurally resemble the Staphylococcus aureus Cna B domain. Each isolated module can bind the plasma glycoprotein asialofetuin as well as the polysaccharide receptors present on the surface of oral streptococci and epithelial cells. Thus, FimA should serve as an excellent paradigm for the development of therapeutic strategies and elucidating the precise molecular mechanisms underlying the interactions between cellular receptors and Gram-positive fimbriae.

The structure of complement C3b provides insights into complement activation and regulation.

The human complement system is an important component of innate immunity. Complement-derived products mediate functions contributing to pathogen killing and elimination. However, inappropriate activation of the system contributes to the pathogenesis of immunological and inflammatory diseases. Complement component 3 (C3) occupies a central position because of the manifold biological activities of its activation fragments, including the major fragment, C3b, which anchors the assembly of convertases effecting C3 and C5 activation. C3 is converted to C3b by proteolysis of its anaphylatoxin domain, by either of two C3 convertases. This activates a stable thioester bond, leading to the covalent attachment of C3b to cell-surface or protein-surface hydroxyl groups through transesterification. The cleavage and activation of C3 exposes binding sites for factors B, H and I, properdin, decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46), complement receptor 1 (CR1, CD35) and viral molecules such as vaccinia virus complement-control protein. C3b associates with these molecules in different configurations and forms complexes mediating the activation, amplification and regulation of the complement response. Structures of C3 and C3c, a fragment derived from the proteolysis of C3b, have revealed a domain configuration, including six macroglobulin domains (MG1-MG6; nomenclature follows ref. 5) arranged in a ring, termed the beta-ring. However, because neither C3 nor C3c is active in complement activation and regulation, questions about function can be answered only through direct observations on C3b. Here we present a structure of C3b that reveals a marked loss of secondary structure in the CUB (for 'complement C1r/C1s, Uegf, Bmp1') domain, which together with the resulting translocation of the thioester domain provides a molecular basis for conformational changes accompanying the conversion of C3 to C3b. The total conformational changes make many proposed ligand-binding sites more accessible and create a cavity that shields target peptide bonds from access by factor I. A covalently bound N-acetyl-l-threonine residue demonstrates the geometry of C3b attachment to surface hydroxyl groups.

Crystallography of gram-positive bacterial adhesins.

Both Gram-negative and Gram-positive pathogens display a multitude of proteins and protein assemblies (pili or fimbriae) on their cell surfaces, which are often used for adherence and initiate colonization and pathogenesis. Adhesive proteins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), anchored by a specific enzyme called sortase in Gram-positive bacteria, target the host's extracellular matrix proteins (ECM) like collagen, fibrinogen and fibronectin. In the past decade, structural analysis by X-ray crystallography has enhanced our understanding of the interactions between MSCRAMMs and the host ECM by revealing several novel structural features that dictate surface protein assembly and the mode of their adhesion to host tissue. The latest focus is on the recently discovered Gram-positive bacterial pili, assembly of which is assisted by yet another specific sortase. Novel features like inter- and intra-molecular isopeptide bonds that facilitate the stability of the pilins, and intra-molecular donor strand complementation to stabilize the adhesin-target interactions are specific to Gram-positive bacteria. This chapter describes and discusses the common structural details between surface proteins and pilins of Gram-positive bacteria and biological implications emanating from these structures.

The crystal structure of cobra venom factor, a cofactor for C3- and C5-convertase CVFBb.

Cobra venom factor (CVF) is a functional analog of human complement component C3b, the active fragment of C3. Similar to C3b, in human and mammalian serum, CVF binds factor B, which is then cleaved by factor D, giving rise to the CVFBb complex that targets the same scissile bond in C3 as the authentic complement convertases C4bC2a and C3bBb. Unlike the latter, CVFBb is a stable complex and an efficient C5 convertase. We solved the crystal structure of CVF, isolated from Naja naja kouthia venom, at 2.6 A resolution. The CVF crystal structure, an intermediate between C3b and C3c, lacks the TED domain and has the CUB domain in an identical position to that seen in C3b. The similarly positioned CUB and slightly displaced C345c domains of CVF could play a vital role in the formation of C3 convertases by providing important primary binding sites for factor B.

Purification, crystallization and halide phasing of a Streptococcus agalactiae backbone pilin GBS80 fragment.

The Gram-positive pathogen Streptococcus agalactiae or group B streptococcus (GBS) is the leading cause of bacterial septicemia, pneumonia and meningitis among neonates around the world. The pathogen assembles two types of pili on its surface, named PI-1 and PI-2, that mediate bacterial adherence to host cells. The GBS PI-1 pilus is formed by the major pilin GBS80, which forms the pilus shaft, and two minor pilins GBS104 and GBS52, which are incorporated into the pilus structure. While considerable structural information exists on Gram-negative pili, the structural study of Gram-positive pili is an emerging area of research. Here, the purification, crystallization and initial phasing of the 35 kDa major fragment of the backbone pilin GBS80 are reported. Crystals were obtained in two different space groups: P2(1) and C2. SAD data collected from an iodide-derivative crystal at the home source were used to obtain initial phases and interpretable electron-density maps.

Preliminary crystallographic study of the Streptococcus agalactiae sortases, sortase A and sortase C1.

Sortases are cysteine transpeptidases that are essential for the assembly and anchoring of cell-surface adhesins in Gram-positive bacteria. In Streptococcus agalactiae (GBS), the pilin-specific sortase SrtC1 catalyzes the polymerization of pilins encoded by pilus island 1 (PI-1) and the housekeeping sortase SrtA is necessary for cell-wall anchoring of the resulting pilus polymers. These sortases are known to utilize different substrates for pilus polymerization and cell-wall anchoring; however, the structural correlates that dictate their substrate specificity have not yet been clearly defined. This report presents the expression, purification and crystallization of SrtC1 (SAG0647) and SrtA (SAG0961) from S. agalactiae strain 2603V/R. The GBS SrtC1 has been crystallized in three crystal forms and the GBS SrtA has been crystallized in one crystal form.

Structural insights into the role of the N-terminus in the activation and function of extracellular serine protease from Staphylococcus epidermidis.

Extracellular serine protease (Esp) from Staphylococcus epidermidis is a glutamyl endopeptidase that inhibits the growth and formation of S. aureus biofilms. Previously, crystal structures of the matured and active Esp have been determined. Interestingly, many of the staphylococcal glutamyl endopeptidase zymogens, including V8 from Staphylococcus aureus and Esp from S. epidermidis, contain unusually long pro-peptide segments; however, their function is not known. With the aim of elucidating the function of these pro-peptide segments, crystal structures of the Esp zymogen (Pro-Esp) and its variants were determined. It was observed that the N-terminus of the Pro-Esp crystal structure is flexible and is not associated with the main body of the enzyme, unlike in the known active Esp structure. In addition, the loops that border the putative substrate-binding pocket of Pro-Esp are flexible and disordered; the structural components that are responsible for enzyme specificity and efficiency in serine proteases are disordered in Pro-Esp. However, the N-terminal locked Pro-Esp variants exhibit a rigid substrate-binding pocket similar to the active Esp structure and regain activity. These structural studies highlight the role of the N-terminus in stabilizing the structural components responsible for the activity and specificity of staphylococcal glutamyl endopeptidases.

Novel structure of the N-terminal helical domain of BibA, a group B streptococcus immunogenic bacterial adhesin.

BibA, a group B streptococcus (GBS) surface protein, has been shown to protect the pathogen from phagocytic killing by sequestering a complement inhibitor: C4b-binding protein (C4BP). Here, the X-ray crystallographic structure of a GBS BibA fragment (BibA126-398) and a low-resolution small-angle X-ray scattering (SAXS) structure of the full-length N-terminal domain (BibA34-400) are described. The BibA126-398 fragment crystal structure displayed a novel and predominantly helical structure. The tertiary arrangement of helices forms four antiparallel three-helix-bundle-motif repeats, with one long helix from a bundle extending into the next. Multiple mutations on recombinant BibA34-400 delayed the degradation of the protein, and circular dichroism spectroscopy of BibA34-400 suggested a similar secondary-structure composition to that observed in the crystallized BibA126-398 fragment. A model was generated for the 92 N-terminal residues (BibA34-125) using structural similarity prediction programs, and a BibA34-400 model was generated by combining the coordinates of BibA34-126 and BibA126-398. The X-ray structure of BibA126-398 and the model of BibA34-400 fitted well into the calculated SAXS envelope. One possible binding site for the BibA N-terminal domain was localized to the N-terminal CCP (complement-control protein) domains of the C4BP α-chain, as indicated by the decreased binding of BibA to a ΔCCP1 C4BP α-chain mutant. In summary, it is suggested that the GBS surface protein BibA, which consists of three antiparallel α-helical-bundle motifs, is unique and belongs to a new class of Gram-positive surface adhesins.

The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation.

The second component of complement (C2) is a multi-domain serine protease that provides catalytic activity for the C3 and C5 convertases of the classical and lectin pathways of human complement. The formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b and the subsequent cleavage of C2 by C1s or MASP2, respectively. The crystal structure of full-length C2 is not yet available, although the structure of its C-terminal catalytic segment C2a has been determined. The crystal structure of the N-terminal segment C2b of C2 determined to 1.8 A resolution presented here reveals the arrangement of its three CCP domains. The domains are arranged differently compared with most other CCP-domain assemblies, but their arrangement is similar to that found in the Ba part of the full-length factor B structure. The crystal structures of C2a, C2b and full-length factor B are used to generate a model for C2 and a discussion of the domain association and possible interactions with C4b during formation of the C4b-C2 complex is presented. The results of this study also suggest that upon cleavage by C1s, C2a domains undergo conformational rotation while bound to C4b and the released C2b domains may remain folded together similar to as observed in the intact protein.

An IgG-like domain in the minor pilin GBS52 of Streptococcus agalactiae mediates lung epithelial cell adhesion.

Streptococcus agalactiae is the leading cause of neonatal pneumonia, sepsis, and meningitis. The pathogen assembles heterotrimeric pilus structures on its surface; however, their function in pathogenesis is poorly understood. We report here the crystal structure of the pilin GBS52, which reveals two IgG-like fold domains, N1 and N2. Each domain is comprised of seven antiparallel beta strands, an arrangement similar to the fold observed in the Staphylococcus aureus adhesin Cna. Consistent with its role as an adhesin, deletion of gbs52 gene significantly reduces bacterial adherence to pulmonary epithelial cells. Moreover, latex beads linked to the GBS52 protein adhere to pulmonary but not to many other epithelial cells; binding to the former is specifically inhibited by antibodies against GBS52. Nonetheless, substantial binding is only observed with N2 domain-conjugated beads. This study presents the structure of a Gram-positive pilin that utilizes a distinct IgG fold variant to mediate pathogen adherence to a specific tissue.

Crystal structure of the N-terminal domain of the fibronectin-binding protein PavA from Streptococcus pneumoniae.

The Gram-positive bacterium Streptococcus pneumoniae, a major human pathogen, is a regular colonizer of the upper and lower respiratory tracts. Pneumococcal adherence and virulence factor A (PavA), a fibronectin-binding bacterial protein, from S. pneumoniae is an important facilitator of its colonization of host cells. In this study, the crystal structure of the N-terminal domain of PavA (SpPavA-N) determined at a resolution of 2.39 Šis reported. Each monomer of the dimeric protein consists of two domains (domains I and II) and a short α-helix (α6) at the C-terminus that are connected by elongated loops. Comparison of the SpPavA-N structure with that of its homolog from Streptococcus suis (FBPS-N) revealed differences in α5, α6 and the domain II/α6 inter-loop region within domain II. The α5 helix of FBPS-N folds back toward domain I, whereas in SpPavA-N it adopts an elongated rod shape.

The crystal structure of C2a, the catalytic fragment of classical pathway C3 and C5 convertase of human complement.

The multi-domain serine protease C2 provides the catalytic activity for the C3 and C5- convertases of the classical and lectin pathways of complement activation. Formation of these convertases requires the Mg(2+)-dependent binding of C2 to C4b, and the subsequent cleavage of C2 by C1s or MASP2, respectively. The C-terminal fragment C2a consisting of a serine protease (SP) and a von Willebrand factor type A (vWFA) domain, remains attached to C4b, forming the C3 convertase, C4b2a. Here, we present the crystal structure of Mg(2+)-bound C2a to 1.9 A resolution in comparison to its homolog Bb, the catalytic subunit of the alternative pathway C3 convertase, C3bBb. Although the overall domain arrangement of C2a is similar to Bb, there are certain structural differences. Unexpectedly, the conformation of the metal ion-dependent adhesion site and the position of the alpha7 helix of the vWFA domain indicate a co-factor-bound or open conformation. The active site of the SP domain is in a zymogen-like inactive conformation. On the basis of these structural features, we suggest a model for the initial steps of C3 convertase assembly.

Identification of novel inhibitors of bacterial surface enzyme Staphylococcus aureus Sortase A.

In-silico virtual screening of bacterial surface enzyme Staphylococcus aureus Sortase A against commercial compound libraries using FlexX software package has led to the identification of novel inhibitors. Inhibition of enzyme catalytic activity was determined by monitoring the steady state cleavage of a model peptide substrate. Preliminary structure activity relationship studies on the lead compound resulted in the identification of compounds with improved activity. The most active compound has an IC50 value of 58 microM against the enzyme.

Deciphering complement mechanisms: the contributions of structural biology.

Since the resolution of the first three-dimensional structure of a complement component in 1980, considerable efforts have been put into the investigation of this system through structural biology techniques, resulting in about a hundred structures deposited in the Protein Data Bank by the beginning of 2007. By revealing its mechanisms at the atomic level, these approaches significantly improve our understanding of complement, opening the way to the rational design of specific inhibitors. This review is co-authored by some of the researchers currently involved in the structural biology of complement and its purpose is to illustrate, through representative examples, how X-ray crystallography and NMR techniques help us decipher the many sophisticated mechanisms that underlie complement functions.

The structure of sortase B, a cysteine transpeptidase that tethers surface protein to the Staphylococcus aureus cell wall.

Many surface proteins of Gram-positive bacteria, which play important roles during the pathogenesis of human infections, are anchored to the cell wall envelope by a mechanism requiring sortases. Sortase B, a cysteine transpeptidase from Staphylococcus aureus, cleaves the C-terminal sorting signal of IsdC at the NPQTN motif and tethers the polypeptide to the pentaglycine cell wall cross-bridge. During catalysis, the active site cysteine of sortase and the cleaved substrate form an acyl intermediate, which is then resolved by the amino group of pentaglycine cross-bridges. We report here the crystal structures of SrtBDeltaN30 in complex with two active site inhibitors, MTSET and E64, and with the cell wall substrate analog tripleglycine. These structures reveal, for the first time, the active site disposition and the unique Cys-Arg catalytic machinery of the cysteine transpeptidase, and they also provide useful information for the future design of anti-infective agents against sortases.