Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: Effect on metal selectivity and activity
Conservative and nonconservative mutations of the transmembrane CPC motif in ZntA: Effect on metal selectivity and activity
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DOI：
10.1021/bi0616394
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发表时间：
2007-03-27
期刊：
影响因子：
2.9
通讯作者：
Mitra, Bharati
中科院分区：
文献类型：
--
作者：
Dutta, Sabari J.;Liu, Junbo;Mitra, Bharati
来自大肠杆菌的ZntA属于P-1B-ATPase转运蛋白家族，介导了对所选二价金属离子毒性水平的抗性。根据底物阳离子选择性的不同，P-1B型ATPase可以分为不同的亚群。ZntA对Pb2+的选择性最高，其次是Zn2+和Cd2+，对Cu2+、Ni2+和Co2+的选择性也较低。它有两个高亲和力的金属结合位点，分别位于N-末端和跨膜结构域。ZntA跨膜金属位点的配体包括第六个跨膜螺旋上保守的(CPC394)-C-392基序的半胱氨酸残基。Pro393在所有P型ATPase中都是不变的。对于具有不同金属离子选择性的锌A同系物，半胱氨酸被丝氨酸、组氨酸和苏氨酸取代。为了测试它们对活性和金属离子选择性的影响，研究了锌锌矿中Cys392或Cys394位的单丙氨酸、组氨酸和丝氨酸的取代，以及组氨酸和丝氨酸对这两种半胱氨酸的双重取代。对P393A进行了表征。C392A、C394A和P393A失去了在跨膜区与高亲和力的金属离子结合的能力。Cys392和Cys394上的组氨酸和丝氨酸替换导致Pb2+在跨膜部位失去结合，这表明CPC基序的两个半胱氨酸都是结合高亲和力的Pb2+所必需的。然而，C392H、C392S、C394H、C394S、C392S/C394S和C392H/C394H可以与其他二价金属离子结合在跨膜位置，并保持较低的但可测量的活性。有趣的是，这些突变体失去了wtZntA所显示的对锌和镉的主要选择性。因此，保守残基提供的配体不仅与金属离子具有高亲和力，而且具有最有利的结合构型，从而导致高效催化，从而有助于金属的选择性。
ZntA from Escherichia coli belongs to the P-1B-ATPase transporter family and mediates resistance to toxic levels of selected divalent metal ions. P-1B-type ATPases can be divided into subgroups based on substrate cation selectivity. ZntA has the highest selectivity for Pb2+, followed by Zn2+ and Cd2+; it also shows low levels of activity with Cu2+, Ni2+, and Co2+. It has two high-affinity metal-binding sites, one each in the N-terminus and the transmembrane domains. Ligands to the transmembrane metal site in ZntA include the cysteine residues of the conserved (CPC394)-C-392 motif in the sixth transmembrane helix. Pro393 is invariant in all P-type ATPases. For ZntA homologues with different metal ion selectivity, the cysteines are replaced by serine, histidine, and threonine. To test the effect on activity and metal ion selectivity, single alanine, histidine, and serine substitutions at Cys392 or Cys394 in ZntA were characterized, as well as double substitutions of both cysteines by histidine or serine. P393A was also characterized. C392A, C394A, and P393A lost the ability to bind a metal ion with high affinity in the transmembrane domain. Histidine and serine substitutions at Cys392 and Cys394 resulted in loss of binding of Pb2+ at the transmembrane site, indicating that both cysteines of the CPC motif are required for binding Pb2+ with high affinity in ZntA homologues. However, C392H, C392S, C394H, C394S, C392S/C394S, and C392H/C394H could bind other divalent metal ions at the transmembrane site and retained low but measurable activity. Interestingly, these mutants lost the predominant selectivity for Zn2+ and Cd2+ shown by wtZntA. Therefore, conserved residues contribute to metal selectivity by supplying ligands that bind metal ions not only with high affinity, as for Pb2+, but also with the most favorable binding geometry that results in efficient catalysis.