Plasminogen Activation, Conformation, and Binding in the Context of Group A Streptococcus Virulence Factors, Streptokinase and PAM
Plasminogen (hPg) conversion to the serine protease plasmin via host and exogenous factors results in activation of the human fibrinolytic system and ultimately degradation of fibrin and extracellular matrices. Group A Streptococcus (GAS), a bacterial pathogen responsible for diseases including rheumatic heart disease, impetigo, and necrotizing fasciitis; activates Pg through the extracellular protein streptokinase (SK). This process is enhanced when virulence factors M or M-like proteins, i.e., PAM, found on the surface of GAS, bind to Pg. Pg has 5 kringle domains (K1-K5), which are homologous triple-disulfide-linked peptide regions of approximately 80 amino acids in length. It has previously been shown that PAM selectively binds to the second kringle domain, K2, of Pg. Kringle domains, excluding K3, bind to ω-amino acids such as lysine and C-terminal lysines.
The work described herein studied the key proteins involved in GAS virulence, specifically Pg, SK, and PAM. Through mutagenesis of the LBS of each kringle it was demonstrated that PAM exclusively binds to the kringle 2 of plasminogen. These hPg mutants all have a faster rate of SK-mediated activation in comparison to WT-hPg. Through studies of isolated kringle 2 and a kringle 2 mutant with the LBS of kringle 1 it was found that the additional Pro in kringle 1 prevents PAM binding due to structural differences. It was also found that SK activates hPg faster when hPg adopts the open conformation; however, the addition of a lysine analogue, EACA, inhibits hPg activation. Lastly, it was found that mPg cannot be activated by SK, but can be activated by the SK-hPg complex. These findings lead to a better mechanistic and structural dynamic of how these three proteins interact.
History
Date Created
2017-07-17Date Modified
2022-10-06Defense Date
2017-07-03Research Director(s)
Francis J. CastellinoCommittee Members
Holly V. Goodson Robert V. StahelinDegree
- Doctor of Philosophy
Degree Level
- Doctoral Dissertation
Program Name
- Chemistry and Biochemistry