The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76–116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50% inhibition at approx. 1µM EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94Å (9.4nm) to 85Å above the phospholipid surface (assuming κ2 = 2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S–C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S–C4b-binding protein with APC on the phospholipid surface. To confirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the α-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the N-terminus of EGF1 of 76Å (κ2 = 2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.
Skip Nav Destination
Follow us on Twitter @Biochem_Journal
Article navigation
August 2000
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
Research Article|
July 25 2000
Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S Available to Purchase
Tilman M. HACKENG;
Tilman M. HACKENG
1
*Departments of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, U.S.A.
†Department of Biochemistry, Cardiovascular Research Institute Maastricht, University Maastricht 6200 MD, The Netherlands
1To whom correspondence should be addressed at University Maastricht (e-mail [email protected]).
Search for other works by this author on:
Subramanian YEGNESWARAN;
Subramanian YEGNESWARAN
*Departments of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, U.S.A.
‡Department of Medical Biochemistry & Genetics, Texas A&M University, Health Science Center and Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Search for other works by this author on:
Arthur E. JOHNSON;
Arthur E. JOHNSON
‡Department of Medical Biochemistry & Genetics, Texas A&M University, Health Science Center and Departments of Chemistry and of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, U.S.A.
Search for other works by this author on:
John H. GRIFFIN
John H. GRIFFIN
*Departments of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, U.S.A.
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
February 29 2000
Accepted:
May 02 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 349 (3): 757–764.
Article history
Received:
February 29 2000
Accepted:
May 02 2000
Citation
Tilman M. HACKENG, Subramanian YEGNESWARAN, Arthur E. JOHNSON, John H. GRIFFIN; Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S. Biochem J 1 August 2000; 349 (3): 757–764. doi: https://doi.org/10.1042/bj3490757
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Biochemical Society Member Sign in
Sign InSign in via your Institution
Sign in via your InstitutionGet Access To This Article
Follow us on Twitter @Biochem_Journal
Open Access for all
We offer compliant routes for all authors from 2025. With library support, there will be no author nor reader charges in 5 journals. Check here |
![]() View past webinars > |