Coiled-coil destabilizing residues in the group A Streptococcus M1 protein are required for functional interaction

Abstract

The sequences of M proteins, the major surface-associated virulence factors of the widespread bacterial pathogen group A Streptococcus, are antigenically variable but have in common a strong propensity to form coiled coils. Paradoxically, these sequences are also replete with coiled-coil destabilizing residues. These features are evident in the irregular coiled-coil structure and thermal instability of M proteins. We present an explanation for this paradox through studies of the B repeats of the medically important M1 protein. The B repeats are required for interaction of M1 with fibrinogen (Fg) and consequent proinflammatory activation. The B repeats sample multiple conformations, including intrinsically disordered, dissociated, as well as two alternate coiled-coil conformations: a Fg-nonbinding register 1 and a Fg-binding register 2. Stabilization of M1 in the Fg-nonbinding register 1 resulted in attenuation of Fg binding as expected, but counterintuitively, so did stabilization in the Fg-binding register 2. Strikingly, these register-stabilized M1 proteins gained the ability to bind Fg when they were destabilized by a chaotrope. These results indicate that M1 stability is antithetical to Fg interaction and that M1 conformational dynamics, as specified by destabilizing residues, are essential for interaction. A "capture-and-collapse" model of association accounts for these observations, in which M1 captures Fg through a dynamic conformation and then collapses into a register 2-coiled coil as a result of stabilization provided by binding energy. Our results support the general conclusion that destabilizing residues are evolutionarily conserved in M proteins to enable functional interactions necessary for pathogenesis.

Keywords: M protein; coiled coil; dynamics; fibrinogen; group A Streptococcus.

Fig. 1.

M1 is a nonideal coiled coil. (A) Schematic of the M1 protein, whose mature form consists of the A region, B repeats, S region, C repeats, and D region. The N-terminal gray region represents the cleaved signal sequence, and the C-terminal gray region the site cleaved by sortase A and attached to the peptidoglycan. (B) Helical wheel and heptad repeat schematic of the two registers of the B repeats of M1 protein. (Upper) register 1 and (Lower) register 2. Coiled-coil destabilizing residues at the a and d positions are circled. The color coding of residues is preserved in the two registers, and the arrows indicate the rotation of the helical face required to transition from one register to the other. The heptad a and d positions are shaded in gray. Residues observed to bind Fg in register 2 are italicized and bolded; the same residues are also italicized and bolded in register 1.

Fig. S1.

Idealization of the B repeats. (A) Heptad positions of residues in the B repeats as predicted by Coils (12). Residues that correspond to register 1 are in red, and those that correspond to register 2 are in blue. (B) Sequence of M1*^1R, with idealizing mutations in black and depicted in register 1. Residues that contact Fg in register 2 are bolded and italicized. (C) Sequence of M1*^2R, with idealizing mutations in black and depicted in register 2. Residues that contact Fg in register 2 are bolded and italicized.

Fig. 2.

Idealized mutants are restricted to the intended register. Formation of intradimer disulfide bonds by (A) AB*^1R and (B) AB*^2R containing 160C (control), 161C (register 2 probe), or 162C (register 1 probe) mutations. (Left, Initial) Proteins were initially heated and reduced and were visualized by Coomassie-stained, nonreducing SDS/PAGE. (Middle, Final) Proteins were then cooled and diluted without reducing agent. The formation of disulfide bonds was assessed by nonreducing SDS/PAGE, which was visualized by Western blot using anti-M1 polyclonal antibodies. (Right, Final) Same as the middle panel, except under reducing conditions.

Fig. 3.

Idealized mutants are more stable than wild-type. CD signal (mean residue ellipticity, MRE) at 4 °C as a function of wavelength (A and C) and at 222 nm as a function of temperature (B and D) of (Upper) AB and (Lower) intact M1 proteins. Wild-type is in blue, register 1-idealized in green, and register 2-idealized in purple.

Fig. 4.

M1 protein samples both registers, but when stabilized in either register, is attenuated in Fg interaction. (A) Disulfide bond formation in wild-type, His-tagged M1 protein containing A160C, L161C (register 2 probe), or E162C (register 1 probe) substitution mutations, as assessed by (Left) nonreducing and (Right) reducing SDS/PAGE and visualized by Western blot using an anti-His antibody. (B) Ni²⁺-NTA agarose coprecipitation assay for interaction of His-tagged AB proteins with FgD at 37 °C. Bound FgD was assessed through Coomassie-stained SDS/PAGE. His-tagged M1 and its bound FgD is included as a reference. (C) Ni²⁺-NTA agarose coprecipitation assay for interaction of His-tagged M1 proteins with FgD at 37 °C. Bound FgD was assessed through Coomassie-stained SDS/PAGE. (D) HBP released in whole blood that was incubated with M1 constructs, as assayed by ELISA. PBS with no bacteria was used as a negative control. Values represent the mean from two donors, with samples from each donor being measured in duplicate; the SD is shown. (E) Ni²⁺-NTA agarose coprecipitation assay for interaction of His-tagged M1 proteins with FgD at 37 °C carried out in the presence of 3.5 M urea. Bound FgD was assessed through Coomassie-stained SDS/PAGE.

Fig. S2.

Intradimer versus interdimer disulfide bond formation. Disulfide bond formation at 10-fold higher (0.5 mg/mL) or the same concentration (0.05 mg/mL) as in Fig. 4A, as assessed by nonreducing SDS/PAGE and visualized by Western blot using an anti-His antibody.

Fig. S3.

Interaction with Fg. (A) Unbound proteins from Ni²⁺-NTA coprecipitation assay for interaction of His-tagged AB proteins with FgD, as shown in Fig. 4B. (B) Unbound proteins from Ni²⁺-NTA coprecipitation assay for interaction of His-tagged M1 proteins with FgD, as shown in Fig. 4C. (C) Ni²⁺-NTA agarose coprecipitation assay for interaction of His-tagged M1 proteins with FgD at 37 °C carried out in the presence of 2 M (Left) or 3 M (Right) urea. Bound FgD was assessed through Coomassie-stained SDS/PAGE. (D) Unbound proteins from Ni²⁺-NTA coprecipitation assay for interaction of His-tagged M1 proteins with FgD, as shown in Fig. 4E.

Fig. S4.

AB*^2R is a structured protein. ¹H-¹⁵N HSQC spectra of (A) AB (B) and AB*^2R collected at 26 °C.

Fig. S5.

Idealized M1 proteins on the GAS surface. (A) Surface expression of M1 protein by wild-type GAS 5448, GAS 5448 (Δemm1) carrying an empty plasmid or plasmids encoding wild-type M1, register 1-stabilized M1, or register 2-stabilized M1, as assayed by FACS, using a polyclonal anti-M1 protein antibody. The values are the means of three triplicates normalized to GAS 5448, with the SD shown. (B) Binding of FITC-labeled Fg to the same strains as in A as assayed by FACS. The values are normalized to the value for GAS 5448, with the SD shown.

Fig. 5.

Capture-and-collapse model of M1–Fg interaction. The B repeats of M1 protein are flexible and sample register 1 (blue), register 2 (red), dissociated, and intrinsically disordered (purple) states. The intrinsically disordered state captures Fg, after which the B repeats collapse into a register 2 coiled coil.