Molecular epitopes of the ankyrin-spectrin interaction
Molecular epitopes of the ankyrin-spectrin interaction
复制标题
DOI：
10.1021/bi702525z
复制
发表时间：
2008-07-15
期刊：
影响因子：
2.9
通讯作者：
MacDonald, Ruby I.
中科院分区：
文献类型：
--
作者：
Ipsaro, Jonathan J.;Huang, Lei;MacDonald, Ruby I.
锚蛋白及其结合伙伴血影蛋白的亚型负责多种人类细胞中的许多相互作用。然而，相互矛盾的证据确定了两个不同的、不重叠的人红系锚蛋白亚结构域 Zu5 和 272，作为 β-血影蛋白的最小结合区域。对血影蛋白锚蛋白结合结构域的补充研究在某种程度上更具结论性，但尚未显示血影蛋白重复序列的定相、整数结合方面的情况。因此，本研究的目的是明确定义和表征最小锚蛋白-血影蛋白结合表位。上述锚蛋白子域的圆二色性 (CD) 波长光谱表明这些片段有 30-60% 是非结构化的。相比之下，人红系β-血影蛋白重复序列13、14、15和16（以两个相邻重复序列的所有组合制备）表现出适当的折叠和稳定性，如CD和色氨酸波长以及热变性扫描所确定的。天然聚丙烯酰胺凝胶电泳 (PAGE) 凝胶位移以及亲和力下拉测定表明 Zu5 和 β-血影蛋白重复序列 14-15 作为最小结合表位。这些结果通过分析超速离心至沉降平衡得到证实，当且仅当 Zu5 与含有串联重复序列 14 和 15 的 β-血影蛋白构建体混合时，才能获得 1:1 复合物。表面等离子体共振产生 β-血影蛋白片段与锚蛋白子结构域 Zu5 结合的 K-D 为 15.2 nM，解释了在完整分子之间观察到的所有结合。总的来说，这些结果表明第 14 个和第 15 个 β-血影蛋白重复序列包含 β-血影蛋白的最小定相区域，它以高亲和力结合 Zu5 亚结构域的锚蛋白。
Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K-D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.