DETERMINANTS OF ION SPECIFICITY ON EF-HANDS SITES - CONVERSION OF THE CA2+/MG2+ SITE OF SMOOTH-MUSCLE MYOSIN REGULATORY LIGHT-CHAIN INTO A CA2+-SPECIFIC SITE
DETERMINANTS OF ION SPECIFICITY ON EF-HANDS SITES - CONVERSION OF THE CA2+/MG2+ SITE OF SMOOTH-MUSCLE MYOSIN REGULATORY LIGHT-CHAIN INTO A CA2+-SPECIFIC SITE
复制标题
DOI：
10.1074/jbc.270.12.6773
复制
发表时间：
1995-03-24
影响因子：
4.8
通讯作者：
REINACH, FC
中科院分区：
文献类型：
--
作者：
DASILVA, ACR;KENDRICKJONES, J;REINACH, FC
钙结合蛋白介导大量的细胞过程。这些过程响应于大量过量镁存在下胞质钙的微摩尔波动。这些蛋白质中存在的金属结合位点是钙特异性的（调节位点）或能够结合钙和镁（结构位点）。使用定点突变，我们能够将鸡平滑肌肌球蛋白调节轻链（RLC）中存在的单个Ca 2 +/Mg 2+位点转化为Ca 2+特异性位点。用谷氨酸替换存在于金属结合环的第12位（-Z配位位置）的天冬氨酸增加了钙亲和力并消除了镁结合，使得该位点具有钙特异性。为了解释这一观察结果，我们假设侧链改变构象的能力的限制，有助于一个（Mg 2+结合）或两个（Ca 2+结合）的协调可能会改变EF手中的金属特异性。其他减少或消除钙结合的突变也已被表征。当用来取代内源性扇贝肌球蛋白RLC时，这些突变体能够恢复肌动蛋白激活的肌球蛋白ATP酶的Ca 2+调节，表明在这些杂合肌球蛋白中，Ca 2+特异性位点（存在于必需轻链上）的调节功能不占据Ca 2 +/Mg 2+位点（存在于调节轻链上）。
Calcium binding proteins mediate a large number of cellular processes. These processes respond to micromolar fluctuations of cytosolic calcium in the presence of a large excess of magnesium. The metal binding sites present in these proteins are either calcium-specific (regulatory sites) or capable of binding both calcium and magnesium (structural sites). Using site-directed mutagenesis we were able to convert the single Ca2+/Mg2+ site present in chicken smooth muscle myosin regulatory light chain (RLC) into a Ca2+-specific site. The replacement of the aspartic acid present in the 12th position (-Z coordinating position) of the metal binding loop with a glutamic acid increases calcium affinity and abolishes magnesium binding, rendering the site calcium-specific. To explain this observation, we hypothesize that restrictions on the ability of side chains to change conformation, contributing one (for Mg2+ binding) or two (for Ca2+ binding) coordinations could alter the metal specificity in EF-hands. Other mutations which decrease or abolish calcium binding have also been characterized. When used to substitute the endogenous scallop myosin RLC, these mutants were capable of restoring the Ca2+ regulation to the actin-activated myosin ATPase demonstrating that in these hybrid myosins, the regulatory function of the Ca2+-specific site (present on the essential light chain) does not the occupancy of the Ca2+/Mg2+ site (present on the regulatory light chain).