Crystal Structure of the Minor Pilin FctB Reveals Determinants of Group A Streptococcal Pilus Anchoring (original) (raw)
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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.
Acta Crystallographica Section D Biological Crystallography, 2013
the pilin-like motif of the minor pilin FctB for linking to the LPXTG motif of the major pilus protein FctA in group A streptococcus (GAS; Linke et al., 2010). Hence, the SpaB subunit can appear along the pilus shaft when the lysine of the (individual or polymerized) SpaA pilin motif is linked to the threonine of the SpaB LAFTG sorting motif by a pilus-specific sortase (Ton-That & Schneewind, 2003; Mandlik et al., 2008), and also as a base for pilus assembly linked to the cell wall (Mandlik et al., 2008). This sortase-mediated pilusassembly mechanism, with some differences, is conserved in several other Gram-positive bacteria, such as GBS, Streptococcus pyogenes (GAS), Streptococcus pneumoniae, Enterococcus faecalis and Actinomyces naeslundii (Kang & Baker, 2012; Krishnan & Narayana, 2011). Three types of pili (type P-1, type P-2a and type P-2b) are encoded by three distinct pilus islands (PI-1, PI-2a and PI-2b) in GBS (
Structural Basis for Group B Streptococcus Pilus 1 Sortases C Regulation and Specificity
PLoS ONE, 2012
Gram-positive bacteria assemble pili through class C sortase enzymes specialized in polymerizing pilin subunits into covalently linked, high-molecular-weight, elongated structures. Here we report the crystal structures of two class C sortases (SrtC1 and SrtC2) from Group B Streptococcus (GBS) Pilus Island 1. The structures show that both sortases are comprised of two domains: an 8-stranded b-barrel catalytic core conserved among all sortase family members and a flexible N-terminal region made of two a-helices followed by a loop, known as the lid, which acts as a pseudo-substrate. In vitro experiments performed with recombinant SrtC enzymes lacking the N-terminal portion demonstrate that this region of the enzyme is dispensable for catalysis but may have key roles in substrate specificity and regulation. Moreover, in vitro FRET-based assays show that the LPXTG motif common to many sortase substrates is not the sole determinant of sortase C specificity during pilin protein recognition.
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.
Infection and Immunity, 2008
Streptococcus [GBS]) infection, a leading cause of neonatal sepsis and meningitis. The genes necessary for pilus synthesis and assembly are clustered in pilus islands (PI). Each gene encodes three structural subunits (a backbone and two ancillary proteins) bearing a C-terminal LPXTG motif and two subfamily C sortases (SrtC) involved in covalent polymerization of the subunits. GBS strains also possess the conserved "housekeeping" sortase A (SrtA), but its role in pilus assembly is unclear. To address this issue, pilus expression and cell wall anchoring were analyzed in srtA deletion mutants. Loss of SrtA did not affect pilus polymerization. However, pilus expression on the cell surface was reduced, and pili accumulated in the culture supernatant. Furthermore, cell-associated pili could be readily released by detergent treatment, indicating that SrtA is involved in covalent anchoring of pili to the cell wall. When each of the genes comprising PI-2a was systematically deleted, only the absence of ancillary subunit GBS150 or the SrtC required for incorporation of GBS150 into pili mimicked the srtA mutant phenotype. Thus, from these data a model for GBS pilus assembly can be proposed in which PI sortases are responsible for polymerization of the pilus structure, while SrtA is required to covalently attach it to the cell wall, utilizing ancillary pilus subunit GBS150 as the anchor protein.
Journal of Biological Chemistry, 2009
The pili expressed by Streptococcus pyogenes and certain other Gram-positive bacterial pathogens are based on a polymeric backbone in which individual pilin subunits are joined end-to-end by covalent isopeptide bonds through the action of sortase enzymes. The crystal structure of the major pilin of S. pyogenes, Spy0128, revealed that each domain of the two domain protein contained an intramolecular isopeptide bond cross-link joining a Lys side chain to an Asn side chain. In the present work, mutagenesis was used to create mutant proteins that lacked either one isopeptide bond (E117A, N168A, and E258A mutants) or both isopeptide bonds (E117A/E258A). Both the thermal stability and proteolytic stability of Spy0128 were severely compromised by loss of the isopeptide bonds. Unfolding experiments, monitored by circular dichroism, revealed a transition temperature T m of 85°C for the wild type protein. In contrast, mutants with only one isopeptide bond showed biphasic unfolding, with the domain lacking an isopeptide bond having a T m that was ϳ30°C lower than the unaltered domain. High resolution crystal structures of the E117A and N168A mutants showed that the loss of an isopeptide bond did not change the overall pilin structure but caused local disturbance of the protein core that was greater for E117A than for N168A. These effects on stability appear also to be important for pilus assembly. The abbreviations used are: GAS, group A streptococcus; ESI-TOF, electrospray ionization-time of flight; WT, wild type; MOPS, 4-morpholinepropanesulfonic acid; N domain, N-terminal domain; C domain, C-terminal domain.
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.
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...
Proceedings of the National Academy of Sciences of the United States of America, 2018
Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA synthesizes pilus...
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...