Variations in the secondary structures of PAM proteins influence their binding affinities to human plasminogen

Abstract

M-proteins (M-Prts) are major virulence determinants of Group A Streptococcus pyogenes (GAS) that are covalently anchored to the cell wall at their conserved COOH-termini while the NH₂-terminal regions extend through the capsule into extracellular space. Functional M-Prts are also secreted and/or released from GAS cells where they exist as helical coiled-coil dimers in solution. Certain GAS strains (Pattern D) uniquely express an M-protein (plasminogen-binding group A streptococcal M-protein; PAM) that directly interacts with human plasminogen (hPg), a process strongly implicated in the virulence of these strains. M-Prt expressed by the emm gene is employed to serotype over 250 known strains of GAS, ∼20 of which are hitherto found to express PAMs. We have developed a modular structural model of the PAM dimer that describes the roles of different domains of this protein in various functions. While the helical COOH-terminal domains of PAM are essential for dimerization in solution, regions of its NH₂-terminal domains also exhibit a weak potential to dimerize. We find that temperature controls the open (unwound) or closed (wound) states of the functional NH₂-terminal domains of PAM. As temperature increases, α-helices are dramatically reduced, which concomitantly destabilizes the helical coiled-coil PAM dimers. PAMs with two a-repeats within the variable NH₂-terminal A-domain (class I/III) bind to hPg tightly, but natural PAM isolates with a single a-repeat in this domain (class II) display dramatic changes in hPg binding with temperature. We conclude that coexistence of two a-repeats in PAM is critical to achieve optimal binding to hPg, especially in its monomeric form, at the biologically relevant temperature.

Keywords: Coiled coils; Dimer dissociation; PAM dimerization; Plasminogen acquisition; Single a-repeat; α-Helix.

Fig. 1.. CD spectra of PAM fragments at different temperatures.

The mean residue ellipticities (MRE; [θ]) of each protein or polypeptide are illustrated as a function of wavelength from 195 nm - 250 nm at the specified temperatures. The results are displayed as data points that represent the average of triplicate scans at a given temperature. The standard error very small at <5% on average for each curve. Panel A represents plots of [θ] vs wavelength for PAM_{AP53_short} at 4° C (black) and 37° C (red). Panel B compares the CD spectra of PAM_{AP53_short} (black) and PAM_{AP53_medium} at 4° C (blue). Panel C illustrates changes in [θ] of full-length PAM_NS88.2 (residues 42-385) vs wavelength at 4° C (black) and 37° C (red). Full length PAMs comprise amino acids immediately downstream of the signal peptide up to, but not including, the sortase A recognition peptide (LPXTG), and hence contain the HVR-A-B-C-D-Pro/Gly domains.

Fig. 2.. Correlation between the fraction of α-helices at 25° C and tolerance to temperature changes.

(A) For each full-length PAM, the x-axis represents the α-helix content at 25° C, and the y-axis represents the ratio of the dimeric content at 37° C/25° C, represented as the fold change between these two temperatures. All data points are labeled as the corresponding PAM, and categorized into 4 clusters in this plot, shown as different colors. (B) The first derivative of the mean residue ellipticity [θ] in the CD temperature scan is plotted against temperature at each point. Curves of different PAMs are shown in distinct colors.

Fig. 3.. Binding assays of PAMs to hPg at 37° C.

hPg was immobilized on a CM-5 chip and titrated with the indicated full-length PAM at the concentrations specified in each panel. (A) For Class I and Class III PAMs, with two a-repeats in the A-domain, kinetic binding analyses are as shown for the example of PAM_NS223 (Class III). Here, SPR response units (RU) were plotted against time and the corresponding K_d values, obtained from k_off/k_on, are provided in Table 5. The experimental data are shown in black lines and fitted curves are shown in red lines. (B) For Class II PAMs, with only a single a-repeat, very high k_off values were observed and were not reliable for calculation, as shown for the example of PAM_SS1448. Here a SPR-based steady state approach was utilized, wherein the highest value of the RU at each concentration was plotted against the PAM_SS1448 concentration, as in (C), and the K_d value was obtained from the calculated concentration midpoint of the titration. The corresponding K_d values are provided in Table 5.

Fig. 4.. Binding assays of PAM-derived peptides to hPg at 25° C.

hPg was immobilized on a CM-5 chip, and peptide concentrations used in the binding assay titrations are indicated in Panels (A)-(D). (A) VEK75_AP53; (B) AGL55_NS88.2, (C) KTI55_SS1448, and (D) VEK75_{AP53_RH1/AA}. Kinetic analyses were used for (A) and (D), and steady state analyses for (B) and (C). In kinetic analyses, the experimental data are shown in black lines and fitted curves are shown in red lines. The corresponding binding constants for these curves are provided in Table 6.

Fig. 5.. Binding assays of PAM-derived peptides to hPg at 15° C and 37° C.

hPg was immobilized on a CM-5 chip and peptide concentrations used for the titrations are indicated in each panel. Kinetic analyses were performed at 15° C for: (A) AGL55_NS88.2, (B) KTI55_SS1448, (C) VEK75_{AP53_RHI/AA}. Steady state analyses were performed at 37° C for (D) AGL55_NS88.2, (E) KTI55_SS1448, (F) VEK75_{AP53_RH1/AA}. In kinetic analyses, the experimental data are shown in black lines and fitted curves are shown in red lines. The corresponding binding constants from both kinetic and affinity analyses are provided in Table 6.

Fig. 6.. Open and closed conformations of the PAM structural model.

Dimeric models were drawn to scale in ChemDraw Professional 16.0 based upon the domain organization of PAM_AP53. (A) At 4° C, the HVR with some α-helix content dimerizes and forms a closed pattern at the NH₂-terminus. (B) At 25° C or 37° C, loss of α-helices within the HVR regions occur, eventually resulting in an open status of NH₂-terminal domains, viz., HVR-A-B domains. The numbers of the first residue in each domain are listed on the illustration.