A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway
A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway
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DOI：
10.1073/pnas.96.15.8384
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发表时间：
1999-07-20
影响因子：
11.1
通讯作者：
Stayton, PS
中科院分区：
文献类型：
--
作者：
Freitag, S;Chu, V;Stayton, PS
目前尚不清楚小分子是沿着定义的途径从蛋白质结合部位解离，还是通过一系列解离途径解离，我们在此报告了一项联合结晶学、计算和生物物理研究，表明Asp-128-->；Ala(D128A)链霉亲和素突变体非常接近于在定义明确的解离途径上的中间体。ASP-128氢键连接到生物素的缩氨基氮上，也与重要的芳香族结合接触点Trp-92和Trp-108网络连接，在D128A结构中，ASN-23与生物素ureido氧的氢键延长到3.8埃，并且一个水分子进入口袋以取代缺失的羧酸盐相互作用，这些变化伴随着生物素的耦合运动，包含Ser-45的柔性结合环和包含Ser-27氢键接触的环。这种结构与在天然链霉亲和素的平均力模拟解离途径中观察到的关键中间体非常相似，在那里ASN-23氢键首先断裂，伴随着Asp-128氢键被进入的水分子取代，此外，生物素和柔性环都在协调的构象变化中移动，这与D128A的结构变化非常相似。测定了D128A突变体的活化参数和热力学参数，与中间体通过吸热键断裂和伴随的构型熵增益穿过解离反应坐标的早期部分相一致。这些综合结果表明，D128A突变体提供了生物素相对定义明确的解离途径上的早期中间体的结构快照。
It is currently unclear whether small molecules dissociate from a protein binding site along a defined pathway or through a collection of dissociation pathways, We report herein a joint crystallographic, computational, and biophysical study that suggests the Asp-128 --> Ala (D128A) streptavidin mutant closely mimics an intermediate on a well-defined dissociation pathway. Asp-128 is hydrogen bonded to a ureido nitrogen of biotin and also networks with the important aromatic binding contacts Trp-92 and Trp-108, The Asn-23 hydrogen bond to the ureido oxygen of biotin is lengthened to 3.8 Angstrom in the D128A structure, and a water molecule has moved into the pocket to replace the missing carboxylate interaction, These alterations are accompanied by the coupled movement of biotin, the flexible binding loop containing Ser-45, and the loop containing the Ser-27 hydrogen bonding contact. This structure closely parallels a key intermediate observed in a potential of mean force-simulated dissociation pathway of native streptavidin, where the Asn-23 hydrogen bond breaks first, accompanied by the replacement of the Asp-128 hydrogen bond by an entering water molecule, Furthermore, both biotin and the flexible loop move in a concerted conformational change that closely approximates the D128A structural changes. The activation and thermodynamic parameters for the D128A mutant were measured and are consistent with an intermediate that has traversed the early portion of the dissociation reaction coordinate through endothermic bond breaking and concomitant gain in configurational entropy. These composite results suggest that the D128A mutant provides a structural "snapshot" of an early intermediate on a relatively well-defined dissociation pathway for biotin.