The fragment 2 domain (F2) of human prothrombin is known to interact with both thrombin and factor Va contributing to enhanced prothrombinase activity in the absence of phospholipid membranes. Using P.Pastoris, we have over-expressed recombinant F2 and achieved uniform labeling of F2 with both 15N and 15N/13C isotopes. The 1H-15N HSQC NMR spectra of F2 and other backbone (13Calpha and some 13C beta) resonances have been assigned by a combination of proton homonuclear TOCSY/NOESY and triple-resonance NMR experiments. The 13C alpha conformational shifts indicate that human F2 assumes secondary structure elements similar to those found in the crystal structure of human F2 in complex with thrombin. Less than stoichiometric amounts of thrombin and factor Va were found to perturb subsets of the HSQC peaks of F2, indicating specific and reversible binding of human F2 with both thrombin and factor Va. The binding sites on F2 for thrombin and factor Va are located at distinct surface areas, as shown through an analysis of residue-specific shifts in the assigned HSQC spectra of F2. Most of the binding residues on F2 identified by NMR correlate well with those identified through functional studies and/or in the crystal structure of the human F2-thrombin complex. More interestingly, our NMR studies demonstrate for the first time direct involvement of the C-terminal region of F2, or the junction residues between kringle 2 and thrombin in prothrombin, for thrombin binding. Therefore, using recombinant F2 we have been able to characterize the thrombin- and Va-binding domains on F2 and establish that F2 binds to these proteins via different binding sites.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.79019 |
| Date | January 2002 |
| Creators | Koutychenko, Anatol |
| Contributors | Ni, Feng (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biochemistry.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001976408, proquestno: AAIMQ88234, Theses scanned by UMI/ProQuest. |
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