Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Av Bento Gonçalves 9500, CP 15005, Porto Alegre 91500-970, RS, Brazil.
Proteins. 2011 Sep;79(9):2735-45. doi: 10.1002/prot.23102. Epub 2011 Jul 18.
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.
抗凝血酶 (AT) 是一种丝氨酸蛋白酶抑制剂，在血液中以两种主要同工型 α 和 β 形式循环，它们在糖基化程度和肝素亲和力方面存在差异。与这种糖胺聚糖结合后，天然 AT 构象，作为蛋白酶抑制剂相对不活跃，被转化为激活形式。在此过程中，β-AT 对肝素表现出更高的亲和力，被认为是体内主要的 AT 糖型抑制剂。然而，无论是证明两种 AT 同工型与肝素结合差异的分子基础，还是其构象激活的机制，都没有得到充分的理解。因此，本工作评估了糖基化和肝素结合对 AT 结构、功能和动力学的影响。基于获得的数据，除了天然和激活形式的 AT 外，还在溶液中自发观察到了一种中间状态，该状态之前被提议存在于这些构象之间。此外，Asn135 连接的寡糖导致 AT 结合的肝素发生弯曲，使多糖远离螺旋 D，从而降低其与 α-AT 的亲和力。获得的数据支持了一个原子水平的、涉及溶剂和氨基酸残基的假设模型的提出，该模型用于从肝素结合外位点到 β-片层 A 和反应中心环传递构象信号，也支持鉴定涉及螺旋 D 的丝氨酸蛋白酶糖型之间的这种传递差异，其中 Asn135 连接的寡糖存在。这些分子内重排，以及肝素在 AT 表面上的动力学，可能为 Asn135 连接的聚糖对肝素结合和 AT 激活的影响提供原子水平的解释。
