Localization and Interaction of Functional Sites on Antithrombin III. Use of an Anti-Hapten Antibody as a Structural Probe

Abstract

Immunochemical and spectral techniques have been used to characterize interaction between functional sites on the protease inhibitor, antithrombin III. Site-directed- modification of a single tryptophan in antithrombin with hydroxynitrobenzyl bromide (HNB) eliminates heparin binding to the protein concomitantly with loss of heparin-promoted inactivation of thrombin by antithrombin. The tryptophan involved in heparin binding is located at position 49 in the sequence, near the N-terminus of antithrombin, and an arginine present at position 393, near the C-terminus, is the site of thrombin binding and inactivation. Although these functional sites are remote in primary sequence, what is their relative orientation in the native three-dimensional structure of the protein? How do these sites communicate with each other? Spectral and haptenic properties of HNB were exploited to answer these questions. In activity measurements, antibody binding to HNB at the heparin binding site at trp-49 did not preclude thrombin inactivation by HNB-antithrombin thrombin. Also, anti-HNB antibodies were shown to bind to the HNB-antithrombin-thrombin complex more avidly than HNB-antithrombin alone, indicating structural changes to occur in the region of trp-49 upon thrombin binding. This immunochemical data was confirmed by spectral measurements on the HNB group, which served as an environmentally sensitive extrinsic probe. Upon binding of thrombin to antithrombin, a shift is observed in the visible HNB spectrum which is consistent with increased exposure of the HNB chromophore to aqueous solvent. These results indicate that: (1) the heparin-binding and thrombin-complexing sites are spatially separated on antithrombin, and (2) the functional regions of antithrombin communicate via conformational signals.