Crystal Structure of the N-terminal Domain of the Group B Streptococcus Alpha C Protein

Abstract

Group B Streptococcus (GBS) is the leading cause of bacterial pneumonia, sepsis, and meningitis among neonates and an important cause of morbidity among pregnant women and immunocompromised adults. Invasive diseases due to GBS are attributed to the ability of the pathogen to translocate across human epithelial surfaces. The alpha C protein (ACP) has been identified as an invasin that plays a role in internalization and translocation of GBS across epithelial cells. The soluble N-terminal domain of ACP (NtACP) blocks the internalization of GBS. We determined the 1.86-Å resolution crystal structure of NtACP comprising residues Ser 52 through Leu 225 of the full-length ACP. NtACP has two domains, an N-terminal ␤-sandwich and a C-terminal three-helix bundle. Structural and topological alignments reveal that the ␤-sandwich shares structural elements with the type III fibronectin fold (FnIII), but includes structural elaborations that make it unique. We have identified a potential integrin-binding motif consisting of Lys-Thr-Asp 146 , Arg 110 , and Asp 118 . A similar arrangement of charged residues has been described in other invasins. ACP shows a heparin binding activity that requires NtACP. We propose a possible heparin-binding site, including one surface of the three-helix bundle, and nearby portions of the sandwich and repeat domains. We have validated this prediction using assays of the heparin binding and cell-adhesion properties of engineered fragments of ACP. This is the first crystal structure of a member of the highly conserved Gram-positive surface alpha-like protein family, and it will enable the internalization mechanism of GBS to be dissected at the atomic level.Group B Streptococcus (GBS) 1 (Streptococcus agalactiae) remains the leading cause of invasive bacterial diseases in neonates, despite its decline in prevalence during the last decade because of intrapartum chemoprophylatic therapy (1, 2). It is also an important cause of morbidity in pregnant women and non-pregnant adults with underlying medical conditions (3-5). GBS colonizes the human gastrointestinal and genitourinary tracts and may cause chorioamnionitis and urinary tract infection in pregnant women and a range of invasive infections in elderly and immunocompromised adults (1, 6 -8). During labor and delivery, GBS may be transmitted to neonates, causing pneumonia, sepsis, or meningitis. Four to six percent of all neonatal GBS infections result in death (6, 9). In vitro, GBS adheres to (10, 11), internalizes within (12-14), and translocates across (15) intact human epithelial and endothelial cells. Little is known about the bacterial components that allow this pathogen to adhere to and penetrate cellular membranes. Previous studies suggest that surface proteins are significantly involved in the process (11, 16). The surface-expressed GBS alpha C protein (ACP) has been shown to act as an invasin (15). ACP is the prototype of a family of surface-expressed proteins containing long tandem repeats (alpha-like proteins (Alp)). Members of this famil...

The group B streptococcal alpha C protein binds α 1 β 1-integrin through a novel KTD motif that promotes internalization of GBS within human epithelial cells

“…RR9 does not affect internalization of either A909 or JL2053 (Bolduc et al, 2002). In addition, structural data suggest that NtACP may bind integrin via a KTD motif in the D1 domain (Auperin et al, 2005). Based on these results and the data presented above, we hypothesize that soluble NtACP may compete for binding to host cell a 1 b 1 -integrin, thus preventing GBS-associated ACP from binding and inhibiting its contribution to the internalization of GBS.…”

“…The amino acid sequence of ACP is in the NCBI Protein Database under accession number AAA26848 (Auperin et al, 2005;Michel et al, 1992). The DNA sequence encoding the ACP (pCL1), NtACP (pDEK14), repeat region RR9 (pET24RR9) and D2R has been cloned previously (Auperin et al, 2005;Bolduc et al, 2002;Gravekamp et al, 1996;Kling et al, 1997).…”

“…The amino acid sequence of ACP is in the NCBI Protein Database under accession number AAA26848 (Auperin et al, 2005;Michel et al, 1992). The DNA sequence encoding the ACP (pCL1), NtACP (pDEK14), repeat region RR9 (pET24RR9) and D2R has been cloned previously (Auperin et al, 2005;Bolduc et al, 2002;Gravekamp et al, 1996;Kling et al, 1997). The aspartic acid residue (Asp 146 ) of the recombinant NtACP was substituted with an alanine residue (D146A) using the Quik-Change site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.…”

“…A close structural homology exists between D1 and FnIII10 (Auperin et al, 2005). In addition to FnIII10's RGD integrin-binding motif, FnIII requires two charged residues (Arg 1379 and Asp 1373 ) located on module 9 (FnIII9) for maximal integrin binding (Hamburger et al, 1999).…”

“…We solved the tertiary structure of NtACP by X-ray crystallography (Auperin et al, 2005). NtACP can be further divided into two structurally distinct domains, D1 and D2.…”