Experimental conditions can obscure the second high-affinity site in LeuT

被引:74
作者
Quick, Matthias [1 ,2 ,3 ]
Shi, Lei [4 ,5 ]
Zehnpfennig, Britta [1 ,2 ]
Weinstein, Harel [4 ,5 ]
Javitch, Jonathan A. [1 ,2 ,3 ,6 ]
机构
[1] Columbia Univ Coll Phys & Surg, Ctr Mol Recognit, New York, NY 10032 USA
[2] Columbia Univ Coll Phys & Surg, Dept Psychiat, New York, NY 10032 USA
[3] New York State Psychiat Inst & Hosp, Div Mol Therapeut, New York, NY 10032 USA
[4] Cornell Univ, Weill Med Coll, Dept Physiol & Biophys, New York, NY 10021 USA
[5] Cornell Univ, Weill Med Coll, HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsau, New York, NY 10021 USA
[6] Columbia Univ Coll Phys & Surg, Dept Pharmacol, New York, NY 10032 USA
基金
美国国家卫生研究院;
关键词
BACTERIAL HOMOLOG; SUBSTRATE; BINDING; MODULATION; SYMPORTER; MECHANISM; PROTEINS; DYNAMICS; NA+;
D O I
10.1038/nsmb.2197
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
NeurotransmitterNa(+) symporters (NSSs), the targets of antidepressants and psychostimulants, recapture neurotransmitters from the synapse in a Na+-dependent symport mechanism. The crystal structure of the NSS homolog LeuT from Aquifex aeolicus revealed one leucine substrate in an occluded, centrally located (S1) binding site next to two Na+ ions. Computational studies combined with binding and flux experiments identified a second substrate (S2) site and a molecular mechanism of Na+-substrate symport that depends upon the allosteric interaction of substrate molecules in the two high-affinity sites. Here we show that the S2 site, which has not yet been identified by crystallographic approaches, can be blocked during preparation of detergent-solubilized LeuT, thereby obscuring its crucial role in Na+-coupled symport. This finding points to the need for caution in selecting experimental environments in which the properties and mechanistic features of membrane proteins can be delineated.
引用
收藏
页码:207 / 211
页数:5
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