An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1

Abstract

The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1's N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N-and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1's elongated stalk and physically "locking" it into place. The structure is stabilized through a highly favorable ΔG solvation on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989-1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.X-ray crystallography | protein folding | adhesin | Streptococcus | intramolecular lock S treptococcus mutans is a recognized cause of human dental caries (cavities), the most common infectious disease worldwide (1). Identifying how S. mutans interacts with host components at the molecular level is essential to fully understand its virulence properties. The sucrose-independent adhesin P1 (AgI/II, antigen B, PAc) is localized on the surface of this oral pathogen, along with many other streptococci (2-7). In the oral cavity, S. mutans P1 interacts with the salivary agglutinin glycoprotein complex composed predominantly of scavenger receptor gp340/DMBT1 (2, 3, 5-10). Without a complete structural model, the mechanisms by which P1 binds to host components have not yet been fully characterized.P1's primary structure (Fig. 1A) contains a 38-residue signal sequence, the heretofore uncharacterized N-terminal region, three alanine-rich repeats (A1-3), a central domain containing a so-called variable (V) region (11), three proline-rich repeats (P1-3), a C-terminal region consisting of three domains (C1-3), an LPxTG sortase-recognition motif, and wall-and membrane-spanning regions (12, 13). Recent partial X-ray crystal structure and velocity centrifugation studies of the intact protein unveiled a unique architecture in which the ∼185-kDa (1,561-aa) protein folds back on itself to form a ∼50-nm elongated hybrid helical stalk that separates two independent adherence domains, with a globular head at the apex and a globular C-terminal region at the base (13-15) (Fig. 1B).The crystal structure of th...

Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c

“…The tertiary structure and cell surface architecture of P1 has been studied extensively [20,[36][37][38][39]. Interestingly, C123, which we demonstrate herein to represent the amyloidforming portion of P1, also associates with covalently attached full-length P1 on the cell surface [39].…”

“…Because an S. mutans sortase-deficient mutant is defective in amyloid formation [19], an additional sortase substrate, GbpC, which was not identified in our initial screen but demonstrated high amyloid prediction scores by computational analyses [34,35], was also evaluated in these experiments. Unlike the proteins listed above, for which little structural information is available at present, the complete tertiary structure of P1 has been modelled based on crystal structures of several partial polypeptides [36][37][38]. Importantly, it is now recognized that the C-terminal region of P1 exists in two separate forms: firstly, in the context of the full-length molecule where it is positioned in proximity to the cell wall, and secondly, as a naturally processed and isolated C123 polypeptide that associates with covalently attached fulllength P1 on the cell surface [39].…”

Structural and Functional Analysis of Cell Wall-anchored Polypeptide Adhesin BspA in Streptococcus agalactiae

“…The stalk is generated by a high affinity interaction between the ␣-helix and polyproline II (PPII)-helices of the A and P domains, respectively, serving to present the V "head" domain at its tip (9). The N terminus forms a stabilizing scaffold by wrapping behind the base of the stalk (10). Crystal structures of the V regions of SpaP and SspB have revealed a common architecture consisting of a lectin-like fold with a putative binding cleft (11,12).…”

Identification of a Supramolecular Functional Architecture of Streptococcus mutans Adhesin P1 on the Bacterial Cell Surface

“…The A-repeats form a long ␣-helix that intimately intertwines into a left-handed supercoiled structure with the helical polyproline P-repeats to form the stalk (45) The C-terminal region is also globular and comprises three structurally related ␤-sandwich domains stabilized by covalent isopeptide bonds (44,47). The crystal structure of the N terminus of P1 in complex with its C-terminal intramolecular binding partner revealed that it forms a novel stabilizing scaffold at the base of the stalk that contributes to the stability and proper folding of the full-length molecule (48). Numerous monoclonal antibodies (mAbs) targeting different domains of P1 have been generated and characterized, providing powerful molecular probes of the highly complex protein structure (6, 49 -51) (see Fig.…”