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.

Keywords: Streptococcus; X-ray crystallography; adhesin; intramolecular lock; protein folding.

Fig. 1.

Schematic representation of the primary and modeled tertiary structure of P1. (A) Primary structure of P1 and location of polypeptides used in this study. (B) Proposed tertiary model of P1 based on velocity centrifugation and crystal structures of A3VP1 and C123 fragments (13). (C) Diagram showing the locations of the two engineered Cla1 sites (circled in red) that added isoleucine and aspartic acid residues to either side of the proline-rich region (20).

Fig. 2.

Multiple views of the N terminus in complex with the C-terminal region of P1 and comparison with the previously published structure of the C terminus of S. mutans AgI/II. (A) Three separate views displaying the interaction of the N terminus with the C-terminal region. The N terminus interacts with the C terminus at the C1/C2 interface (amino acids 1114–1158) and immediately upstream of the C2/C3 interface (amino acids 1321–1323). This segment continues past the C1 domain to form a previously unidentified fold that wraps behind the base of P1’s hybrid helical stalk. The N terminus is shown in red, the alanine-rich region is in purple, the proline-rich and immediate downstream region is in green, and each domain of the C terminus is in a different shade of blue. Calcium ions identified within the structure are shown in yellow. Magnesium ions are shown in green. (B) Space-filling model displaying the interface of the N terminus (red) with the C-terminal domains. (C) Structure of the C123 domains within the NA1/P3C complex (blue) compared with the previously published C terminus (gold) of S. mutans Ag I/II (13) superimposed with an rmsd of 0.52 Å.

Fig. 3.

Illustration of the stabilizing bonds, represented by dotted black lines, between residues 989–1001 of P1’s postproline-rich region and the N-terminal intramolecular scaffold. The N terminus is shown in red, the alanine-rich region is in purple, and the postproline-rich region is in green. Side chains not participating in stabilizing interactions have been deleted for clarity. (A) Top portion of the N-terminal scaffold, stabilizing residues 989–994 of the postproline region. (B) Bottom portion of the N-terminal scaffold, stabilizing residues 995–1001 of the postproline-rich region.

Fig. 4.

A 2Fo-Fc map contoured at 2σ, displaying the observed electron density of the postproline-rich region and the N terminus. The N terminus is shown in red, the alanine-rich region is in purple, and the postproline-rich region is in green. (A) The postproline-rich region displays a high number of stabilizing bonds with the N-terminal intramolecular scaffold. The orientation of HFHYFK residues (amino acids 992–997) with side chains lying in alternating directions within the “eye” of the N-terminal topological loop are illustrated. (B) The N-terminal scaffold intimately interacts with and wraps behind the postproline-rich region.

Fig. 5.

Thermal stability and refolding after thermal denaturation of rP1-Cla1_Upstream and rP1-Cla1_{Up/Downstream} compared with rP1. (A) The melting curves of rP1-Cla1_Upstream, rP1-Cla1_{Up/Downstream}, and full-length rP1 during thermal denaturation as measured by DSC. (B and C) Changes in circular dichroism spectra during thermal denaturation of rP1-Cla1_Upstream compared with rP1 (B) and rP1-Cla1_{Up/Downstream} compared with rP1 (C). Far-UV CD spectra were measured from 260 to 185 nm over a temperature range of 25–70 °C. Representative spectra are shown at specific temperatures during thermal denaturation. (D) Circular dichroism evaluation of refolded rP1-Cla1_Upstream and rP1-Cla1_{Up/Downstream} compared with rP1 after thermal denaturation. Far-UV CD spectra were measured from 260 to 185 nm at 25 °C to compare the polypeptides with and without previous thermal denaturation at 70 °C.

Fig. 6.

Illustrations of the regions within the tertiary structure of P1 affected by two different mutations that exhibit instability, impeded folding, and inability to adhere to immobilized salivary agglutinin when expressed in S. mutans (17). The N terminus is shown in red, the alanine-rich region is in purple, and the postproline-rich region is in green. (A) The region deleted within the previously characterized NR7 N-terminal deletion polypeptide (31) is highlighted in cyan and includes the intramolecular scaffold formed around the postproline-rich region. (B) The two added isoleucine and aspartic acid residues encoded by the engineered downstream Cla1 site within polypeptide PC967 (20) correspond to positions 999 and 1000 and are highlighted in yellow. The insertion of extra residues at this location apparently perturbs the alignment and subsequent interactions between postproline-rich region residues and the N-terminal intramolecular scaffold.