Intramolecular Isopeptide Bonds Give Thermodynamic and Proteolytic Stability to the Major Pilin Protein of Streptococcus pyogenes (original) (raw)
Related papers
Journal of Biological Chemistry, 2010
Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-Å resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.
Acta crystallographica. Section D, Biological crystallography, 2014
The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to giv...
Journal of Molecular Biology, 2011
The Gram-positive pathogen Streptococcus agalactiae, known as group B streptococcus (GBS), is the leading cause of bacterial septicemia, pneumonia, and meningitis among neonates. GBS assemble two types of pili, PI-1 and PI-2, on their surface to adhere to host cells and initiate colonization for pathogenesis. The GBS PI-1 pilus is made of one major pilin, GBS80, which forms the pilus shaft, and two secondary pilins (GBS104 and GBS52), which are incorporated into the pilus at various places. We report here the crystal structure of the 35 kDa C-terminal fragment from GBS80, which is composed of two IgG-like domains (N2-N3). The structure was solved by the single-wavelength anomalous dispersion (SAD) method using sodium iodide (NaI) soaked crystals and diffraction data collected at the home source. The N2 domain exhibits a cnaA/DEv-IgG fold with two calcium binding sites, while the N3 domain displays a cnaB/IgG-rev fold. We have built a model for full-length GBS80 (N1, N2 and N3) with the help of available homologous major pilin structures, and we propose a model for the GBS PI-1 pilus shaft. The N2 and N3 domains are arranged in tandem along the pilus shaft, whereas the respective N1 domain is tilted by approximately 20° away from the pilus axis. We have also identified a pilin-like motif in the minor pilin GBS52, which might aid its incorporation at the pilus base. * R merge = Σ hkl Σ j |I(hkl) i -[I(hkl)]|/Σ hkl Σ i I(hkl), where I(hkl) are the intensities of symmetry-related reflections and [I(hkl)] is the average intensity over all observations J Mol Biol. Author manuscript; available in PMC 2012 April 15.
European Biophysics Journal, 2010
Adhesion of the serotype M1 Streptococcus pyogenes strain SF370 to human tonsil explants and cultured keratinocytes requires extended polymeric surface structures called pili. In this important human pathogen, pili are assembled from three protein subunits: Spy0125, Spy0128 and Spy0130 through the action of sortase enzymes. For this study, the structural properties of these pili proteins have been investigated in solution. Spy0125 and Spy0128 display characteristics of globular, folded proteins. Circular dichroism suggests a largely b-sheet composition for Spy0128 and Spy0125; Spy0130 appears to contain little secondary structure. Each of the proteins adopts a monodisperse, monomeric state in solution as assessed by analytical ultracentrifugation. Further, smallangle X-ray scattering curves for Spy0125, Spy0128 and Spy0130 suggest each protein adopts an elongated shape, likely comprised of two domains, with similar maximal dimensions. Based on the scattering data, dummy atom models of each of the pili subunits have been reconstructed ab initio. This study provides the first insights into the structure of Streptococcus pyogenes minor pili subunits, and possible implications for protein function are discussed.
Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilusrelated C sortases remain poorly characterized to date, and their mechanisms of transpeptidation and regulation need to be further investigated. The available 3-dimensional structures of these enzymes reveal a typical sortase fold, except for the presence of a unique feature represented by an N-terminal highly flexible loop known as the "lid." This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an autoinhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work, we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme.-Cozzi, R., Zerbini, F., Assfalg, M., D'Onofrio, M., Biagini, M., Martinelli, M., Nuccitelli, A., Norais, N., Telford, J. L., Maione, D., Rinaudo, C. D. Group B Streptococcus pilus sortase regulation: a single mutation in the lid region induces pilin protein polymerization in vitro. FASEB J. 27, 3144 -3154 (2013). www.fasebj.org Key Words: transpeptidation ⅐ backbone protein ⅐ limited proteolysis ⅐ thermal stability ⅐ NMR spectroscopy 1 These authors contributed equally to this work.
Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae
The Journal of biological chemistry, 2017
Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions including attachment, twitching motility, biofilm formation and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting t...
ABSTRACTMany species of pathogenic gram-positive bacteria display covalently crosslinked protein polymers (called pili or fimbriae) that mediate microbial adhesion to host tissues. These structures are assembled by pilus-specific sortase enzymes that join the pilin components together via lysine-isopeptide bonds. The archetypal SpaA pilus fromCorynebacterium diphtheriaeis built by theCdSrtA pilus-specific sortase, which crosslinks lysine residues within the SpaA and SpaB pilins to build the shaft and base of the pilus, respectively. Here, we show thatCdSrtA crosslinks SpaB to SpaA via a K139(SpaB)-T494(SpaA) lysine-isopeptide bond. Despite sharing only limited sequence homology, an NMR structure of SpaB reveals striking similarities with the N-terminal domain of SpaA (NSpaA) that is also crosslinked byCdSrtA. In particular, both pilins contain similarly positioned reactive lysine residues and adjacent disordered AB loops that are predicted to be involved in the recently proposed “la...
Assembly and role of pili in group B streptococci
Molecular Microbiology, 2006
is the leading cause of neonatal pneumonia, sepsis and meningitis. An in silico genome analysis indicated that GBS strain NEM316 encodes five putative sortases, including the major class A sortase enzyme and four class C sortases. The genes encoding the class C sortases are tandemly arranged in two different loci, srtC1-C2 and srtC3-C4 , with a similar genetic organization and are thought to be involved in pilus biosynthesis. Each pair of sortase genes is flanked by LPXTG protein encoding genes, two upstream and one downstream, and a divergently transcribed regulatory gene located upstream from this locus. We demonstrated that strain NEM316 expresses only the srtC3-C4 locus, which encodes three surface proteins (Gbs1474, Gbs1477 and Gbs1478) that polymerize to form appendages resembling pili. Structural and functional analysis of this locus revealed that: (i) the transcriptional activator RogB is required for expression of the srtC3-C4 operon; (ii) Gbs1477, and either SrtC3 or SrtC4 are absolutely required for pilus biogenesis; and (iii) GBS NEM316 pili are composed of three surface proteins, Gbs1477, the bona fide pilin which is the major component, Gbs1474, a minor associated component, and Gbs1478, a pilus-associated adhesin. Surprisingly, pilus-like structures can be formed in the absence of the two minor components, i.e. the puta-tive anchor Gbs1474 or the adhesin Gbs1478. Adherence assays showed that Gbs1478 confers adhesive capacity to the pilus. This study provides the first evidence that adhesive pili are also present in Grampositive pathogens.
Biochemical and Biophysical Research Communications, 2011
Streptococcus pneumoniae type 2 pili are recently identified fimbrial structures extending from the bacterial surface and formed by polymers of the structural protein PitB. Intramolecular isopeptide bonds are a characteristic of the related pilus backbone protein Spy0128 of group A streptococci. Based on the identification of conserved residues in PitB, we predicted two intramolecular isopeptide bonds in PitB. Using a combination of tandem mass spectrometry and Edman sequencing, we show that these bonds were formed between Lys 63-Asn 214 and Lys 243-Asn 372 in PitB. Mutant proteins lacking the intramolecular isopeptide bonds retained the proteolytic stability observed with the wild type protein. However, absence of these bonds substantially decreased the melting temperature of the PitB-derivatives, indicating a stabilizing function of these bonds in PitB of the pneumococcal type 2 pilus.
Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein
PLOS ONE, 2015
Group B Streptococcus (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the Nterminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knockout strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.