title: Effects of glycosylation on heparin binding and antithrombin activation by heparin.
abstract: Antithrombin (AT), a serine protease inhibitor, circulates in blood in two major isoforms, α and β, which differ in their amount of glycosylation and affinity for heparin. After binding to this glycosaminoglycan, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. In this process, β-AT presents the higher affinity for heparin, being suggested as the major AT glycoform inhibitor in vivo. However, either the molecular basis demonstrating the differences in heparin binding to both AT isoforms or the mechanism of its conformational activation are not fully understood. Thus, the present work evaluated the effects of glycosylation and heparin binding on AT structure, function, and dynamics. Based on the obtained data, besides the native and activated forms of AT, an intermediate state, previously proposed to exist between such conformations, was also spontaneously observed in solution. Additionally, Asn135-linked oligosaccharide caused a bending in AT-bounded heparin, moving such polysaccharide away from helix D, which supports its reduced affinity for α-AT. The obtained data supported the proposal of an atomic-level, solvent and amino acid residues accounting, putative model for the transmission of the conformational signal from heparin binding exosite to β-sheet A and the reactive center loop, also supporting the identification of differences in such transmission between the serpin glycoforms involving helix D, where the Asn135-linked oligosaccharide stands. Such intramolecular rearrangements, together with heparin dynamics over AT surface, may support an atomic-level explanation for the Asn135-linked glycan influence over heparin binding and AT activation.
authors.affiliation: Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av Bento Gonçalves 9500, CP 15005, Porto Alegre 91500-970, RS, Brazil.
authors.forename: Laercio
authors.initials: L
authors.lastname: Pol-Fachin
authors.forename: Camila
authors.initials: C
authors.lastname: Franco Becker
authors.forename: Jorge
authors.initials: J
authors.lastname: Almeida Guimarães
authors.forename: Hugo
authors.initials: H
authors.lastname: Verli
mesh_terms: D000990:Antithrombin III / Q000737:chemistry* / Q000378:metabolism*; D001216:Asparagine; D001665:Binding Sites; D002236:Carbohydrate Conformation; D018360:Crystallography, X-Ray; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D018583:Pliability; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms
chemical_list: D020033:Protein Isoforms; D001216:Asparagine; D000990:Antithrombin III; D006493:Heparin