Search

The therapeutic action of tissue plasminogen activator (t-PA) is a two-step process: (1) binding to lysine-rich fibrin (Km event) and (2) converting local plasminogen into plasmin (Kcat event). Overcoming limitations of other structural biophysics methods, we wanted to employ small-angle X-ray scattering (SAXS) to visualize what shape changes occur/accompany t-PA activation, but the prime hurdle was the polydisperse nature of the fibrin, which occluded scattering information from t-PA. Earlier, larger polylysine peptides have been used to potentiate activation of t-PA, so while screening short polylysine peptides as alternatives to fibrin or larger peptides, we found that penta-polylysine (P5) specifically activates t-PA in a dose-dependent manner, averaging to almost 3-fold more than in the absence of any peptide. SAXS data analysis confirmed that P5 does not induce association of t-PA molecules, and a narrower peak profile of the Kratky plot indicated that P5 binding quenches inherent motion in t-PA. Shape reconstruction of t-PA ∓ P5 revealed that P5 closes the "gap" between the two gross domains of t-PA, viz. fused F/E, K1 and K2 domains, and the P domain. Docking experiments suggested that, while other polylysine peptides preferentially interacted with the surfaces of kringle domains, P5 "slipped into" the gap/groove between K2 and P domains, thereby mediating a substantial increase in the number of long-range interactions between the K2 domain and exosites in the P domain. We report here dissection of shape events involved in between Km/Kcat steps of t-PA activation, which can pave the way toward the search for small molecule function regulator(s) of t-PA.

Human tissue-plasminogen activator (t-PA) is a multidomain glycoprotein which holds high biomedical value due to its therapeutic role in clot-specific fibrinolysis. Although atomic-resolution structures of individual domains except Kringle1 are available, no structural information is available on how these domains and glycosylation are oriented in space relative to each other in the full-length protein. SAXS intensity profile acquired from samples of t-PA was used to "steer" structures of individual domains and the homology model of the first kringle domain to generate a structural model of the protein part of t-PA. Differences in the shape profiles of SAXS data-based dummy atom and proteinogenic models aided in grafting glycosylated moieties on the coordinates of t-PA. According to previously reported mutagenesis-rendered altered functional profiles, normal-mode analysis of our model revealed that the fibrin binding F/E domains "communicate" with the active-site in the P domain via Kringle2, while Kringle1 is positioned away from these long-distance interactions.