Inactivation of the lysine binding sites of human plasminogen (hPg) reveals novel structural requirements for the tight hPg conformation, M-protein binding, and rapid activation

Abstract

Accelerated activation of the human plasminogen zymogen (hPg) to two-chain active plasmin (hPm) is achieved following conformational changes induced by ligand-binding at the lysine-binding sites (LBSs) in four of the five hPg kringle domains. In this manner, pattern D skin-trophic strains of Group A streptococci (GAS), through the expression of surface plasminogen-binding M-protein (PAM), immobilize surface hPg, thereby enabling rapid hPg activation by GAS-secreted streptokinase (SK). Consequently, GAS enhances virulence by digesting extracellular and tight cellular junctional barriers using hPm activity. Many studies have demonstrated the singular importance of the kringle-2 domain of hPg (K2hPg) to PAM-binding using hPg fragments. Recently, we showed, using full-length hPg, that K2hPg is critical for PAM binding. However, these studies did not eliminate any modulatory effects of the non-K2hPg LBS on this interaction. Moreover, we sought to establish the significance of the intramolecular interaction between Asp219 of the LBS of K2hPg and its serine protease domain binding partner, Lys708, to conformational changes in hPg. In the current study, selective inactivation of the LBS of K1hPg, K4hPg, and K5hPg revealed that the LBS of these kringle domains are dispensable for hPg binding to PAM. However, the attendant conformational change upon inactivation of K4hPg LBS increased the affinity of hPg for PAM by an order of magnitude. This finding suggests that the native hPg conformation encloses PAM-binding exosites or sterically hinders access to K2hPg. While simultaneous inactivation of the LBS of K1hPg, K4hPg, and K5hPg inhibited hPg/SK association alongside hPg activation, the replacement of Lys708 generated a slight conformational change that optimally accelerated hPg activation. Thus, we accentuate disparate functions of hPg LBS and conclude, using intact proteins, that K2hPg plays a central role in regulating hPg activation.

Streptolysin O Accelerates the Conversion of Plasminogen to Plasmin

“…In addition, we observe an exosite for SLO-binding like what has been reported for SKA or PAM interaction with PLG. 53 The significant reduction of intra-links found within PLM compared to PLG indicates that there is a drastic conformation change upon formation of PLM that may separate the primary and secondary binding sites (exosite) in space, leading to a loss of SLO-binding as evidenced by the ELISA and the XL-MS results…”

A high-resolution cryo-EM structure of a bacterial M-protein reveals a compact structure that diverges from related M-proteins

“…While we could not resolve hPg to the same high degree as that of PAMAP53 (Supplementary Fig. S3), we nonetheless were able to map the known region of hPg (8,11,13,(33)(34)(35) that was bound to PAM by placing the 3D structure of the hPg-K2 domain within the confines of the experimentally-determined map coordinates.…”

Plasminogen missense variants and their involvement in cardiovascular and inflammatory disease

“…Thus, specific PLG/plasmin cellular receptors are needed. In mammalian cells, glycolytic moonlighting proteins, such as enolase, play important roles in this regard ( 19 , 28 – 32 ), whereas in microbial cells, surface proteins, such as M-protein, and even enolase, which migrates from the cytoplasm to the cell surface by an unknown mechanism, are important PLG receptors used by bacteria for migration and dissemination ( 33 ).…”

“…Likewise, in the LBS of PLG-K2, Asp 219 , Glu 221 , and Arg 234 , interact with other residues located in the SP domain of PLG. These interactions serve to place PLG in a tightly folded and closed activation-resistant T-conformation ( 32 , 38 – 40 ), thus maintaining PLG in plasma, which otherwise would be activated, with the resulting plasmin rapidly inactivated by circulating inhibitors. Upon binding to cellular receptors via the LBS, the intramolecular interactions between the LBS' and the AP and SP residues are displaced, inducing a change that relaxes the conformation of the bound PLG (R) rendering it highly activatable ( 41 ).…”

“…Most recently, systematic inactivation of critical LBS residues in the various kringle domains of PLG was used to determine their effects on PLG activation by tPA, uPA, or SK. The results indicated that the LBS of PLG-K2 has the highest influence on relaxing the PLG conformation and enhancing its activation potential, followed by PLG-K4 and PLG-K5, with PLG-K1 having the smallest influence ( 32 ).…”