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The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin.

ABSTRACT: Antithrombin (AT) is the most important inhibitor of coagulation proteases. Its activity is stimulated by glycosaminoglycans, such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and in the presence of pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from beta-sheet A, which is known to be critical for the allosteric activation of AT. Here, we present data that reveal an energetically distinct intermediate on the path to full activation where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native-like state in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulfide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free-energy contribution of conformational change is provided by the final step of hinge-region expulsion and subsequent closure of the main beta-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in beta-sheet A.

SUBMITTER: Langdown J

PROVIDER: S-EPMC2697666 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin.

Langdown Jonathan J Belzar Klara J KJ Savory Wendy J WJ Baglin Trevor P TP Huntington James A JA

Journal of molecular biology 20090301 5

Antithrombin (AT) is the most important inhibitor of coagulation proteases. Its activity is stimulated by glycosaminoglycans, such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and in the presence of pentasaccharide. Pentasaccha ...[more]

PMID: 19452598

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Project description:The present study deals with the conformation in solution of two heparin octasaccharides containing the pentasaccharide sequence GlcN(NAc,6S)-GlcA-GlcN(NS,3,6S)-IdoA(2S)-GlcN(NS,6S) [AGA*IA; where GlcN(NAc,6S) is N-acetylated, 6-O-sulfated alpha-D-glucosamine, GlcN(NS,3,6S) is N,3,6-O-trisulfated alpha-D-glucosamine and IdoA(2S) is 2-O-sulfated IdoA (alpha-L-iduronic acid)] located at different positions in the heparin chain and focuses on establishing geometries of IdoA residues (IdoA(2S) and IdoA) both inside and outside the AGA*IA sequence. AGA*IA constitutes the active site for AT (antithrombin) and is essential for the expression of high anticoagulant and antithrombotic activities. Analysis of NMR parameters [NOEs (nuclear Overhauser effects), transferred NOEs and coupling constants] for the two octasaccharides indicated that between the 1C4 and 2S0 conformations present in dynamic equilibrium in the free state for the IdoA(2S) residue within AGA*IA, AT selects the 2S0 form, as previously shown [Hricovini, Guerrini, Bisio, Torri, Petitou and Casu (2001) Biochem. J. 359, 265-272]. Notably, the 2S0 conformation is also adopted by the non-sulfated IdoA residue preceding AGA*IA that, in the absence of AT, adopts predominantly the 1C4 form. These results further support the concept that heparin-binding proteins influence the conformational equilibrium of iduronic acid residues that are directly or indirectly involved in binding and select one of their equi-energetic conformations for best fitting in the complex. The complete reversal of an iduronic acid conformation preferred in the free state is also demonstrated for the first time. Preliminary docking studies provided information on the octasaccharide binding location agreeing most closely with the experimental data. These results suggest a possible biological role for the non-sulfated IdoA residue preceding AGA*IA, previously thought not to influence the AT-binding properties of the pentasaccharide. Thus, for each AT binding sequence longer than AGA*IA, the interactions with the protein could differ and give to each heparin fragment a specific biological response.

Project description:Essentials Heparin-binding site (HBS) variants of antithrombin (AT) are associated with thrombosis risk. HSB variants have, in general, normal progressive inhibitory activity but reduced heparin affinity. Thrombosis in HSB carriers has been primarily attributed to the loss of heparin cofactor activity. Results here demonstrate that HSB variants of AT also lack anti-inflammatory signaling functions. SUMMARY:Background Several heparin-binding site (HBS) variants of antithrombin (AT) have been identified that predispose carriers to a higher incidence of thrombosis. Thrombosis in carriers of HBS variants has been primarily attributed to a loss in their heparin-dependent anticoagulant function. Objective The objective of this study was to determine whether HSB mutations affect the anti-inflammatory functions of variants. Methods Two HBS variants of AT (AT-I7N and AT-L99F), which are known to be associated with a higher incidence of thrombosis, were expressed in mammalian cells and purified to homogeneity. These variants were characterized by kinetic assays followed by analysis of their activities in established cellular and/or in vivo inflammatory models. The possible effects of mutations on AT structure were also evaluated by molecular modeling. Results The results indicated that, whereas progressive inhibitory activities of variants were minimally affected, their heparin affinity and inhibitory activity in the presence of heparin were markedly decreased. Unlike wild-type AT, neither AT variant was capable of inhibiting activation of nuclear factor-?B or downregulation of expression of cell adhesion molecules in response to lipopolysaccharide (LPS). Similarly, neither variant elicited barrier protective activity in response to LPS. Structural analysis suggested that the L99F substitution locally destabilizes AT structure. Conclusions It is concluded that the L99F mutation of AT is associated with destabilization of the serpin structure, and that the loss of anti-inflammatory signaling function of the HBS variants may also contribute to enhanced thrombosis in carriers of HBS mutations.

Dataset Information

The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin.

Publications

The critical role of hinge-region expulsion in the induced-fit heparin binding mechanism of antithrombin.

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