Structure and Function of Pattern D Streptococcus Pyogenes Surface Exposed Plasminogen Receptors

One important process governing the pathogenesis of Group A Streptococcus (GAS) involves its conscription of human host plasminogen (hPg) by a multicopy surface-resident M-protein (M-Prt) direct binding hPg receptor (PAM), or other surface moonlighting proteins such as the homooctameric glycolytic enzyme enolase (SEN). hPg is a single chain multi-modular zymogen containing five kringle domains (K1-K5), four of which interact with lysine. This characteristic is utilized by hPg/hPm to interact with receptors containing COOH-terminal lysine residues or through-space isosteric pseudo lysine residues. On the GAS exterior, hPg is readily activated to the serine protease, plasmin (hPm), by bacterial and host activators. Enabling such a proteolytic surface on GAS aids in its survival and dissemination. The ectodomains of evolutionary-related M-Prts are assumed to form extended alpha-helical coiled-coil projections without tertiary structure, while SEN has no features that suggest the mechanism of its translocation from its normal location in the cytoplasm to the cell surface. In this thesis, there are two conclusions drawn for these Pg receptors (PgRs). First, PAM is anchored in membranes of secreted microvesicular (MV) projections both on the GAS surface and in the surrounding medium where it is fully capable of binding hPg/hPm. Secondly, SEN is also found to translocate to the cell surface by lipid microvesicles (MV) most likely originating from the cytosolic membrane. Using cryogenic-electron microscopy, a high-resolution structure of PAM on lentiviral membrane, SEN, and SEN-hPg embedded in small DOPG unilamellar vesicles. (PL), is provided as a model system for MV-PgRs. We found that the ectodomain of PAM in complex with hPg is folded through intra- and inter-domain interactions to a more compact form on the cell surface, thus establishing a new paradigm for membrane-bound M-Prt/ligand structures. While the PL-SEN structure predominantly shows that two subunits of the SEN octamer are exposed to the extracellular medium and six subunits are buried within the membrane and vesicular interior. While soluble SEN does not meaningfully interact with hPg, due to the steric unavailability of the COOH-terminal lysine residue of SEn, hPg can interact with SEN in DOPG vesicles, primarily via the hPg-K1 lysine binding site. Nonetheless, SEN does not place hPg in a favorable conformation for facile activation and this lack of conformational rearrangement is the primary reason for the attenuated enhancement of hPg activation when bound to the SEN octamer. These studies provide a molecular framework for addressing the need for new treatments of GAS disease by providing a platform to solve structures of virulence-determining membrane proteins.