The Signature 3-O-Sulfo Group of the Anticoagulant Heparin Sequence Is Critical for Heparin Binding to Antithrombin but Is Not Required for Allosteric Activation*
AUTOR(ES)
Richard, Benjamin
FONTE
American Society for Biochemistry and Molecular Biology
RESUMO
Heparin and heparan sulfate glycosaminoglycans allosterically activate the serpin, antithrombin, by binding through a specific pentasaccharide sequence containing a critical 3-O-sulfo group. To elucidate the role of the 3-O-sulfo group in the activation mechanism, we compared the effects of deleting the 3-O-sulfo group or mutating the Lys114 binding partner of this group on antithrombin-pentasaccharide interactions by equilibrium binding and rapid kinetic analyses. Binding studies over a wide range of ionic strength and pH showed that loss of the 3-O-sulfo group caused a massive ∼60% loss in binding energy for the antithrombin-pentasaccharide interaction due to the disruption of a cooperative network of ionic and nonionic interactions. Despite this affinity loss, the 3-O-desulfonated pentasaccharide retained the ability to induce tryptophan fluorescence changes and to enhance factor Xa reactivity in antithrombin, indicative of normal conformational activation. Rapid kinetic studies showed that loss of the 3-O-sulfo group affected both the ability of the pentasaccharide to recognize native antithrombin and its ability to preferentially bind and stabilize activated antithrombin. By contrast, mutation of Lys114 solely affected the preferential interaction of the pentasaccharide with activated antithrombin. These findings demonstrate that the 3-O-sulfo group functions as a key determinant of heparin pentasaccharide activation of antithrombin both by contributing to the Lys114-independent recognition of native antithrombin and by triggering a Lys114-dependent induced fit interaction with activated antithrombin that locks the serpin in the activated state.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2785635Documentos Relacionados
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