Contributions of different modules of the plasminogen-binding Streptococcus pyogenes M-protein that mediate its functional dimerization

Abstract

Group A Streptococcus pyogenes (GAS) is a causative agent of pharyngeal and dermal infections in humans. A major virulence determinant of GAS is its dimeric signature fibrillar M-protein (M-Prt), which is evolutionarily designed in modules, ranging from a hypervariable extracellular N-terminal region to a progressively more highly conserved C-terminus that is covalently anchored to the cell wall. Of the >250 GAS isolates classified, only the subset of skin-trophic Pattern D strains expresses a specific serotype of M-Prt, PAM, that directly binds to host human plasminogen (hPg) via its extracellular NH-terminal variable A-domain region. This interaction allows these GAS strains to accumulate components of the host fibrinolytic system on their surfaces to serve extracellular functions. While structure-function studies have been accomplished on M-Prts from Pattern A-C GAS isolates with different direct ligand binding properties compared to PAM, much less is known regarding the structure-function relationships of PAM-type M-Prts, particularly their dimerization determinants. To examine these questions, PAMs from seven GAS strains with sequence variations in the NH-terminal ligand binding domains, as well as truncated versions of PAM, were designed and studied. The results from bioinformatic and biophysical analyses show that the different domains of PAM are disparately engaged in dimerization. From these data, we propose an experimentally-based model for PAM secondary and quaternary structures that is highly dependent on the conserved helical C-terminal C-D-domains. In addition, while the N-terminal regions of PAMs are variable in sequence, the binding properties of hPg and its activated product, plasmin, to the A-domain, remain intact.

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

“…Lastly, a sortase A recognition site and a single pass-through membrane spanning region containing a short cytoplasmic tail make up the emm gene product (Fischetti et al, 1988). Processing at the COOH-terminal sortase A site anchors PAM to the cell wall, after which the fibrous rod-like protein projects through the outer capsule into the extracellular medium, wherein the majority of the protein is available for interactions with the host (Fischetti, 1989;Phillips et al, 1981b;Qiu et al, 2018;Sanderson-Smith et al, 2008). Variations in M-Prts consist of differing numbers and sequences of short homologous repeat sequences within the major domains (Fischetti, 1989;Smeesters et al, 2010).…”

“…We have previously cloned and expressed the extracellular regions of PAMs from serologically distinct Pattern D GAS isolates (Qiu et al, 2018). As with other M-Prts, PAMs exist in solution as coiled-coil dimers (Cedervall et al, 1997;McNamara et al, 2008;Stewart et al, 2016), and we have constructed a structural model that depicts the manner in which PAMs form non-ideal dimers in solution (Qiu et al, 2018). The exposed NH 2terminal A-domain is responsible for specific binding of PAM to the lysine binding site of the ~80-residue kringle 2 (K2 hPg ) domain of the multi-modular hPg (Castellino and Ploplis, 2003).…”

Toward Better Understanding on How Group AStreptococcusManipulates Human Fibrinolytic System

“…In bacterial strains such as PAM NS265 and PAM NS32 , the second RH motif is mutated to Arg-Tyr and Gly-His, respectively (Figure 5a). Despite these variations, all PAM bind to human Plg with high affinities [71,73,74].…”

“…Based on NMR studies [74], the structure of the HVR and A domain is predominantly disordered, and the binding to Plg results in a major conformational change and formation of α-helical structures (Figure 5b). This observation was further supported by experimental data published in a recent study [75], where it is revealed that the conformation switch can be detected even without binding to Plg, and the alternation between disordered and a dimeric α-helical structure occurs in a temperature-dependent manner, similar to the M1 protein reported previously [76].…”

“…Other than the aforementioned dynamic and dimeric interaction at the N-terminal HVR and A domains, the current structure model of PAM is a coiledcoil dimer, which is stabilized via the C and D domains' interaction [74]. It is proposed that at least two C domains are required for a stable dimer formation.…”