Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively

The collagen binding integrin α2β1 plays a crucial role in hemostasis, fibrosis, and cancer progression amongst others. It is specifically inhibited by rhodocetin (RC), a C-type lectin-related protein (CLRP) found in Malayan pit viper (Calloselasma rhodostoma) venom. The structure of RC alone reveals a heterotetramer arranged as an αβ and γδ subunit in a cruciform shape. RC specifically binds to the collagen binding A-domain of the integrin α2 subunit, thereby blocking collagen-induced platelet aggregation. However, until now, the molecular basis for this interaction has remained unclear. Here, we present the molecular structure of the RCγδ-α2A complex solved to 3.0 Å resolution. Our findings show that RC undergoes a dramatic structural reorganization upon binding to α2β1 integrin. Besides the release of the nonbinding RCαβ tandem, the RCγ subunit interacts with loop 2 of the α2A domain as result of a dramatic conformational change. The RCδ subunit contacts the integrin α2A domain in the “closed” conformation through its helix C. Combined with epitope-mapped antibodies, conformationally locked α2A domain mutants, point mutations within the α2A loop 2, and chemical modifications of the purified toxin protein, this molecular structure of RCγδ-α2A complex explains the inhibitory mechanism and specificity of RC for α2β1 integrin.胶原结合型整合素α2β1（collagen binding integrin α2β1）在止血、纤维化及癌症进展等多种生理病理过程中发挥关键作用。该整合素可被鳄毒素（rhodocetin, RC）特异性抑制——RC是一种存在于马来亚竹叶青蛇（Calloselasma rhodostoma）毒液中的C型凝集素相关蛋白（C-type lectin-related protein, CLRP）。单独解析的RC结构显示，其以异四聚体形式存在，由αβ与γδ亚基组装而成，整体呈十字形构象。RC可特异性结合整合素α2亚基的胶原结合A结构域，进而阻断胶原诱导的血小板聚集。然而截至目前，二者相互作用的分子基础仍未明确。本研究解析了分辨率为3.0 Å的RCγδ-α2A复合物分子结构。研究结果表明，RC在结合α2β1整合素后会发生显著的结构重排：一方面无结合活性的RCαβ亚基二聚体发生解离，另一方面RCγ亚基通过剧烈的构象变化与α2A结构域的环2（loop 2）产生相互作用；RCδ亚基则通过其C螺旋与处于“闭合”构象的整合素α2A结构域发生接触。结合表位定位抗体、构象锁定型α2A结构域突变体、α2A环2内的点突变以及纯化毒素蛋白的化学修饰实验，RCγδ-α2A复合物的分子结构阐明了RC对α2β1整合素的抑制机制及其特异性。