Crystal Structures of Multicopper Oxidase CueO Bound to Copper(I) and Silver(I) FUNCTIONAL ROLE OF A METHIONINE-RICH SEQUENCE
Crystal Structures of Multicopper Oxidase CueO Bound to Copper(I) and Silver(I) FUNCTIONAL ROLE OF A METHIONINE-RICH SEQUENCE
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DOI：
10.1074/jbc.m111.293589
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发表时间：
2011-10-28
影响因子：
4.8
通讯作者：
Montfort, William R.
中科院分区：
文献类型：
--
作者：
Singh, Satish K.;Roberts, Sue A.;Montfort, William R.
多铜氧化酶CueO氧化有毒的Cu（I），并且是大肠杆菌中铜稳态所需的。像许多参与铜稳态的蛋白质一样，CueO具有被认为对铜处理至关重要的富含甲硫氨酸的片段。人们对这些片段的功能知之甚少。在这里，我们报告的晶体结构的CueO在1.1埃与45个残基的蛋氨酸丰富的部分完全解决，揭示了一个N-末端螺旋段与蛋氨酸残基并列的铜（I）连接和C-末端高度移动的段富含蛋氨酸和组氨酸残基。我们还报告了CueO的结构与C500 S突变，这导致T1铜的损失，和CueO与六个蛋氨酸改变为丝氨酸。用Cu（I）浸泡C500 S CueO晶体，或用Ag（I）浸泡野生型CueO晶体，导致占据三个位点，即先前确定的底物结合位点和沿富含甲硫氨酸的螺旋沿着的两个新位点，涉及甲硫氨酸358、362、368和376。这些残基的突变导致Cu（I）氧化的k（cat）类似于4倍的降低。Ag（I），其在自然界中通常与铜一起出现，在体外强烈抑制CueO氧化酶活性，并且在体内损害铜耐受性，特别是在缺乏互补铜流出cus系统的情况下。总之，这些研究证明了CueO的富含甲硫氨酸的插入物在Cu（I）的结合和氧化中的作用，并突出了cue和cus系统在铜和银稳态中的相互作用。
The multicopper oxidase CueO oxidizes toxic Cu(I) and is required for copper homeostasis in Escherichia coli. Like many proteins involved in copper homeostasis, CueO has a methionine-rich segment that is thought to be critical for copper handling. How such segments function is poorly understood. Here, we report the crystal structure of CueO at 1.1 angstrom with the 45-residue methionine-rich segment fully resolved, revealing an N-terminal helical segment with methionine residues juxtaposed for Cu(I) ligation and a C-terminal highly mobile segment rich in methionine and histidine residues. We also report structures of CueO with a C500S mutation, which leads to loss of the T1 copper, and CueO with six methionines changed to serine. Soaking C500S CueO crystals with Cu(I), or wild-type CueO crystals with Ag(I), leads to occupancy of three sites, the previously identified substrate-binding site and two new sites along the methionine-rich helix, involving methionines 358, 362, 368, and 376. Mutation of these residues leads to a similar to 4-fold reduction in k(cat) for Cu(I) oxidation. Ag(I), which often appears with copper in nature, strongly inhibits CueO oxidase activities in vitro and compromises copper tolerance in vivo, particularly in the absence of the complementary copper efflux cus system. Together, these studies demonstrate a role for the methionine-rich insert of CueO in the binding and oxidation of Cu(I) and highlight the interplay among cue and cus systems in copper and silver homeostasis.