Speicher D W, DeSilva T M, Speicher K D, Ursitti J A, Hembach P, Weglarz L
Wistar Institute for Anatomy and Biology, Philadelphia, Pennsylvania 19104.
J Biol Chem. 1993 Feb 25;268(6):4227-35.
Head-to-head association of two spectrin alpha beta heterodimers to form tetramers involves the formation of two equivalent alpha-beta complexes. The sites on the alpha subunit N-terminal region and beta subunit C-terminal region that form these alpha beta complexes have been identified using protease footprinting and direct binding assays. The existence of a similar previously hypothesized internal head-to-head alpha beta interaction in dimers was also demonstrated. The discrete regions of both subunits that are protected from proteolysis in tetramers and dimers are not due to the laterally associated subunit since head-to-head complexes of a univalent alpha peptide with a univalent beta peptide show similar protection of the same sites. These sites are unshielded immediately after monomers assemble side-to-side to form heterodimers, demonstrating that reconstituted dimers are initially in an "open" conformation. Conversion of open dimers to a closed form through formation of the internal head-to-head alpha beta association, as demonstrated by restoration of protease protection, occurred on a time scale of hours at 0 degrees C. Analysis of peptide binding affinities as well as isolation and sequence analysis of head-to-head alpha beta noncovalent complexes further defined the regions required for association on both subunits. These regions are homologous to the 106-residue repetitive motif that comprises most of both chains. An algorithm designed to improve prediction accuracy of multiple homologous motifs was used to model the conformation of spectrin repetitive motifs as well as the contact regions. In this model, the separate alpha and beta binding sites are incomplete complementary parts of a triple stranded folding unit. Formation of the alpha beta head-to-head complex produces a triple stranded conformational unit that is slightly different from other homologous motifs in the protein. Most hemolytic anemia mutations that are known to disrupt tetramer association are located in the mapped regions, including several mutations that induce a conformational change in the paired subunit.
两个血影蛋白αβ异源二聚体头对头结合形成四聚体涉及两个等效α-β复合物的形成。利用蛋白酶足迹法和直接结合试验已确定了α亚基N端区域和β亚基C端区域上形成这些α-β复合物的位点。还证实了二聚体中先前假设的类似内部头对头α-β相互作用的存在。在四聚体和二聚体中受蛋白水解保护的两个亚基的离散区域并非由于侧向结合的亚基，因为单价α肽与单价β肽的头对头复合物对相同位点显示出类似的保护。这些位点在单体并排组装形成异源二聚体后立即未被屏蔽，表明重构的二聚体最初处于“开放”构象。如蛋白酶保护的恢复所示，通过形成内部头对头α-β缔合将开放二聚体转化为封闭形式，在0℃下需数小时。对肽结合亲和力的分析以及头对头α-β非共价复合物的分离和序列分析进一步确定了两个亚基上缔合所需的区域。这些区域与构成两条链大部分的106个残基重复基序同源。使用一种旨在提高多个同源基序预测准确性的算法对血影蛋白重复基序的构象以及接触区域进行建模。在该模型中，单独的α和β结合位点是三链折叠单元的不完整互补部分。α-β头对头复合物的形成产生了一个三链构象单元，该单元与蛋白质中其他同源基序略有不同。已知破坏四聚体缔合的大多数溶血性贫血突变位于映射区域，包括几个在配对亚基中诱导构象变化的突变。
