The
            <i>Corynebacterium diphtheriae</i>
            shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Kang, Hae Joo; Paterson, Neil G.; Gaspar, Andrew H.; Ton‐That, Hung; Baker, Edward N.

doi:10.1073/pnas.0906826106

Cited by 104 publications

(174 citation statements)

References 40 publications

Supporting

Mentioning

166

Contrasting

Order By: Relevance

“…For example, the folded SpaA pilin from C. diphtheriae measures 8.0 nm from the C terminus in the crystal structure, at Lys484, to the interpilin cross-link, at Lys190, where force propagates to the next subunit in the pilus (19) (Fig. 1D).…”

Section: Resultsmentioning

confidence: 99%

“…1C). With a 2.48-Å metalligand bond distance, Kang et al (19) postulated the presence of one Ca 2+ ion, despite the lack of Ca 2+ in the crystallization buffer. We hypothesized that mechanical unfolding may disrupt the Ca 2+ -binding site of SpaA, releasing the metal ion into solution, where it would be sequestered by EDTA in the sample buffer.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Pathogenic bacteria adhere despite severe mechanical perturbations induced by the host, such as coughing. In Gram-positive bacteria, extracellular protein appendages termed pili are necessary for adherence under mechanical stress. However, little is known about the behavior of Gram-positive pili under force. Here, we demonstrate a mechanism by which Gram-positive pili are able to dissipate mechanical energy through mechanical unfolding and refolding of isopeptide bond-delimited polypeptide loops present in Ig-type CnaA domains. Using single-molecule force spectroscopy, we find that these loops of the pilus subunit SpaA of the SpaA-type pilus from Corynebacterium diphtheriae and FimA of the type 2 pilus from Actinomyces oris unfold and extend at forces that are the highest yet reported for globular proteins. Loop refolding is limited by the hydrophobic collapse of the polypeptide and occurs in milliseconds. Remarkably, both SpaA and FimA initially refold to mechanically weaker intermediates that recover strength with time or ligand binding. Based on the high force extensibility, CnaA-containing pili can dissipate ∼28-fold as much energy compared with their inextensible counterparts before reaching forces sufficient to cleave covalent bonds. We propose that efficient mechanical energy dissipation is key for sustained bacterial attachment against mechanical perturbations.bacterial adhesion | mechanical stability | single-molecule force spectroscopy | Gram-positive pili | isopeptide bond B acterial infections of solid tissues begin with the attachment of bacteria to target surfaces. In many instances, bacteria adhere against forces that oppose such attachment: micturition in the genitourinary tract (1) or mucociliary flow in the respiratory tract (2), for example. In such environments, a completely immobile adherent bacterium experiences a drag force that can be approximated by Stokes law, F = 6·π·r·η·v, where r is the Stokes radius of the bacterium (∼0.5 μm), η is the viscosity of the fluid (in the respiratory mucus, 1-100 Pa·s −1 ) (3), and v is the velocity of the fluid surrounding the bacterium (Fig. 1A). Under normal mucociliary flow (1-100 μm·s −1 ) (4), forces on a single bacterium can exceed several nanonewtons. Such high forces are sufficient to cleave covalent bonds within the initial adherence structures (5), which would terminate attachment. Understanding how bacteria manage to remain attached under such strong mechanical perturbations is of fundamental interest and could identify new targets for antibiotic development.The initial interaction between bacteria and the host is mediated by micrometer-long adhesive structures termed pili or fimbriae (Fig. 1A). Due to their adhesive role, pili are virulence factors that contribute to the development of infections (6). Structurally, pili are polymers of tens to hundreds of subunits, termed shaft pilins, that are assembled in series and are presented at the extracellular surface, often with inclusion of minor pilins that can have adhesive properties (6). Remar...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Echelman

Alegre-Cebollada

Badilla

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The surface of the C-terminal domains are comprised of an even mix of polar (44%) and non-polar (56%) residues (as calculated by Naccess 2.1.1). Each of these domains also carries an isopeptide bond, a characteristic signature present in Gram-positive pili proteins (25,26) that are typically coordinated through 3 principal residues, lysine, asparagine, and aspartate. Within the C 1 , C 2 , and C 3 domains, the isopeptide bonds link the residues Lys-1006 -Asn-1121, Lys-1161-Asn-1311, and Lys-1338 -Asn-1473, respectively.…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

Larson

Rajashankar

Crowley

et al. 2011

Journal of Biological Chemistry

126

View full text Add to dashboard Cite

The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 Å resolution crystal structure of the complete C-terminal region of AgI/ II. The C-terminal region is comprised of three major domains: C 1 , C 2 , and C 3 . Each domain adopts a DE-variant IgG fold, with two ␤-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C 1 and C 2 ) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C 1 and C 2 domains. Competition experiments demonstrated that the C-and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C 1 and C 2 domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.Dental caries (also called tooth decay or dental cavities) is a ubiquitous worldwide disease that affects humans of all age groups. Streptococcus mutans, a primary etiological agent of human dental caries (1) and an increasingly recognized cause of bacterial endocarditis (2), adheres to proteins contained within the salivary pellicle on the tooth surface, the extracellular matrix, and other microbial species (3). Antigen I/II (AgI/II, 2 also known as P1, B, SpaP, or PAc) of S. mutans has been implicated in bacterial adherence to constituents of the salivary pellicle (4, 5) and has been studied for the past three decades as a target for protective immunity against dental caries. Apart from adherence, AgI/II influences biofilm formation (6) and promotes platelet aggregation (7), collagen-dependent bacterial invasion of dentin (8), and cariogenicity (9). Although AgI/II was initially discovered on oral streptococci, it has also been identified in members of the Group A and Group B streptococci (10), suggesting a role for this adhesin in a variety of species.The AgI/II family proteins range from 140 to 180 kDa in predicted size and have a primary sequence composed of multiple conserved regions (Fig. 1b). Toward the N terminus, repeated sequences of high alanine content constitute the alanine-rich region followed by a segment commonly referred to as the variable (V) region. Further C-terminal in the sequence is a region of high proline content that forms a repetitive prolinerich region. Following the AgI/II proline-rich region is a C-terminal region (60 kDa or 550 amino acids), which is the most conserved region of AgI/II, with 62% identity ...

show abstract

“…The enzymatic reaction involves a nucleophilic cysteine residue, which is essential for the covalent intermolecular association between pilus subunits (13)(14)(15). In addition, intramolecular bonds within pilin subunits, formed between lysine and asparagine side chains, have been identified in the high resolution structures of major pilins Spy0128 from S. pyogenes (16) and SpaA from C. diphtheriae (17) as well as in the main adhesin of the Streptococcus pneumoniae pilus, RrgA (18). The role of these unusual intramolecular cross-links in pilin subunit stabilization has been shown by site-specific mutagenesis and both proteolytic and thermal stability studies (19,20).…”

mentioning

confidence: 99%

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Mortaji

Terrasse

Dessen

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Pili are surface-exposed virulence factors involved in bacterial adhesion to host cells. The Streptococcus pneumoniae pilus is composed of three structural proteins, RrgA, RrgB, and RrgC and three transpeptidase enzymes, sortases SrtC-1, SrtC-2, and SrtC-3. To gain insights into the mechanism of pilus formation we have exploited biochemical approaches using recombinant proteins expressed in Escherichia coli. Using site-directed mutagenesis, mass spectrometry, limited proteolysis, and thermal stability measurements, we have identified isopeptide bonds in RrgB and RrgC and demonstrate their role in protein stabilization. Co-expression in E. coli of RrgB together with RrgC and SrtC-1 leads to the formation of a covalent RrgB-RrgC complex. Inactivation of SrtC-1 by mutation of the active site cysteine impairs RrgB-RrgC complex formation, indicating that the association between RrgB and RrgC is specifically catalyzed by SrtC-1. Mass spectrometry analyses performed on purified samples of the RrgB-RrgC complex show that the complex has 1:1 stoichiometry. The deletion of the IPQTG RrgB sorting signal, but not the corresponding sequence in RrgC, abolishes complex formation, indicating that SrtC-1 recognizes exclusively the sorting motif of RrgB. Finally, we show that the intramolecular bonds that stabilize RrgB may play a role in its efficient recognition by SrtC-1. The development of a methodology to generate covalent pilin complexes in vitro, facilitating the study of sortase specificity and the importance of isopeptide bond formation for pilus biogenesis, provide key information toward the understanding of this complex macromolecular process.

show abstract

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Cited by 104 publications

References 40 publications

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

CnaA domains in bacterial pili are efficient dissipaters of large mechanical shocks

Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

Stability and Assembly of Pilus Subunits of Streptococcus pneumoniae

Contact Info

Product

Resources

About