Perez Eduardo, West Ann H, Stock Ann M, Djordjevic Snezana
Center for Advanced Biotechnology and Medicine, Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Biochemistry. 2004 Feb 3;43(4):953-61. doi: 10.1021/bi035455q.
Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.
细菌趋化性受体在其细胞质结构域内通过特定谷氨酸残基的羧基甲基化进行翻译后修饰。这种高度调控的可逆修饰抵消了配体结合的信号效应，并有助于适应性调节。基于γ-谷氨酰甲基转移酶CheR的晶体结构，我们推测该酶N端结构域α2螺旋中的带正电荷残基可能与甲基转移酶底物（细菌趋化性受体）带负电荷的甲基化区域互补。构建了几种改变后的CheR蛋白，其中带正电荷的精氨酸或赖氨酸残基被丙氨酸取代，并检测它们对野生型受体和一系列含有不同可用于甲基化的谷氨酸的受体变体的甲基化活性。一种CheR突变蛋白（Arg53Ala）对所有受体构建体的活性显著降低，这表明Arg53可能在甲基转移催化中起普遍作用。其余突变蛋白对不同受体底物表现出不同的相对甲基化率模式，表明存在特异性，可能是通过CheR与受体在甲基化特定位点远端的位点相互作用实现的。这些发现意味着CheR的α2螺旋带正电荷残基与受体带负电荷的谷氨酸之间存在互补性。这种互补性很可能参与区分受体的不同甲基化状态。
