Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of alpha- and PPII-helices

Abstract

Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein adhesin that interacts with salivary components within the salivary pellicle. AgI/II contributes to virulence and has been studied as an immunological and structural target, but a fundamental understanding of its underlying architecture has been lacking. Here we report a high-resolution (1.8 A) crystal structure of the A(3)VP(1) fragment of S. mutans AgI/II that demonstrates a unique fibrillar form (155 A) through the interaction of two noncontiguous regions in the primary sequence. The A(3) repeat of the alanine-rich domain adopts an extended alpha-helix that intertwines with the P(1) repeat polyproline type II (PPII) helix to form a highly extended stalk-like structure heretofore unseen in prokaryotic or eukaryotic protein structures. Velocity sedimentation studies indicate that full-length AgI/II that contains three A/P repeats extends over 50 nanometers in length. Isothermal titration calorimetry revealed that the high-affinity association between the A(3) and P(1) helices is enthalpically driven. Two distinct binding sites on AgI/II to the host receptor salivary agglutinin (SAG) were identified by surface plasmon resonance (SPR). The current crystal structure reveals that AgI/II family proteins are extended fibrillar structures with the number of alanine- and proline-rich repeats determining their length.

Fig. 1.

(A) The primary sequence layout of AgI/II is shown at the Top. The alanine-rich repeats are highlighted in shades of blue, and the proline-rich repeats are in shades of purple. Shown Below are the fragments of AgI/II used in this study, including the crystallized fragment A₃VP₁. Residue numbers correspond to the primary sequence of AgI/II from S. mutans strain NG8 (GenBank accession GQ456171). (B) The sequence alignments of A₁, A₂, and A₃ repeats, where the 19 heptad motifs are highlighted in red. (C) The alignments of P₁, P₂, and P₃ repeats, where the proline residues are shown in red.

Fig. 2.

Antigen I/II layout and A₃VP₁ structure overview. (A) Ribbon diagram of the crystal structure of A₃VP₁ generated by PyMol (48). The α-helix from A₃ and the PPII helix from the P₁ regions interact to form the 155 Å stalk. (B) Electrostatic map of A₃VP₁ generated using MolMol (49). The stalk formed by the association of the A₃ and P₁ helices displays an extensive hydrophobic surface. (C) Superposition of the unique molecules from the P2₁ and P2₁2₁2₁ crystal forms by strictly anchoring over the V region shows ~17 Å displacement at the termini of the helices.

Fig. 3.

Interactions between the A₃ and P₁ helices. (A) The helical wheel diagram (Top) shows the interaction between the A₃’s heptad motif “AxYxAx[LV]” and P₁’s “PxxP” motif. The helical net of the interactions between the α- and PPII helices is also shown, where the A₃-repeat heptad motifs are highlighted in blue and the P₁ repeat PxxP motifs are highlighted in orange. Asparagine residues within the A₃ region, intervening the heptads, that are involved in hydrogen bonding with the P₁ PPII helix are highlighted in red. The conserved tyrosines and leucines (highlighted in yellow) of the heptad sequences are nestled between prolines of the P repeat. Additionally, the phenolic oxygen atom of the tyrosine residues participate in water-mediated hydrogen bonding (red-dashed lines). (B) The stereo diagram of the heptad interactions shows that tyrosine side chains nestle between the prolines in a knobs-in-holes interaction, which is highlighted by surface plots for the A and P repeats. (C) The stereo diagram of the region intervening the heptads shows a dominant direct hydrogen bonding between the asparagine side chains of A₃ and the main chain oxygen and nitrogen of the PPII helix. Two prolines (Pro855 and Pro858) break this pattern and face outward.

Fig. 4.

Calorimetry, ELISA, and BIACore studies. (A) Calorimetry of the A/P interaction. (Upper) Calorimetric measurements at 20 °C, 25 injections of 10 μL A_1–3 into the cell containing VP₃. (Lower) The energy (kcal/mol) released during each injection. (B) Inhibition of binding of anti-AgI/II MAbs to S. mutans whole cells. Twofold serial dilutions of AgI/II polypeptides (5–0.05 μM) were incubated with each indicated MAb. Competition ELISA results are shown as the percent inhibition of binding of the indicated MAb to S. mutans whole cells in the presence of each polypeptide. Error bars indicate standard deviations. CG14 (yellow squares), A₃VP₁ (red diamonds), A₁VP₃ (blue squares), and C-terminal construct (green triangles). (C) BIAcore studies of AgI/II fragments with immobilized SAG. Full-length CG14, A₃VP₁, C-terminal, or V-region fragments (analytes) were flowed over a CM5 chip coated with covalently attached SAG. Analyte proteins ranged from 0.25 μM to 4 μM concentrations. Arrows indicate injection starting and stopping points. During the 4-min injection of each polypeptide, an increase in the RU is observed as AgI/II polypeptides bind to SAG. CG14 and A₃VP₁ showed the maximal binding responses (1 RU is equivalent to 1 pg per square millimeter of sensor surface).

Fig. 5.

Model of the AgI/II structure and predicted binding with SAG. (A) The crystal structure of the AgI/II A₃VP₁ region revealed an extended stalk formed by the A₃ and P₁ repeats. Ultracentrifugation studies predict that the A₂–P₂ and A₁–P₃ repeats extend the stalk to a length over 50 nm. (B) Adherence studies indicated the presence of two sites, one within A₃VP₁ and another within the C-terminal region, which are widely separated in the AgI/II structure. The cartoon represents a possible model for AgI/II binding to SAG, where interactions occur at both the distal end through the A₃VP₁ region, and at a secondary adherence site mediated by the C-terminal domain.