Savage David F, Stroud Robert M
Graduate Group in Biophysics, University of California at San Francisco, San Francisco, CA 94158, USA.
J Mol Biol. 2007 May 4;368(3):607-17. doi: 10.1016/j.jmb.2007.02.070. Epub 2007 Mar 2.
The aquaporin family of channels was defined based on the inhibition of water transport by mercurial compounds. Despite the important role of mercurials, little is known about the structural changes involved upon mercury binding leading to channel inhibition. To elucidate the mechanism we designed a mutant, T183C, of aquaporin Z (AqpZ) patterned after the known mercury-sensitive site of aquaporin 1 (AQP1) and determined the X-ray crystal structures of the unbound and mercury blocked states. Superposition of the two structures shows no conformational rearrangement upon mercury binding. In the blocked structure, there are two mercury sites, one bound to Cys183 and occluding the pore, and a second, also bound to the same cysteine but found buried in an interstitial cavity. To test the mechanism of blockade we designed a different mutant, L170C, to produce a more effective mercury block at the pore site. In a dose-response inhibition study, this mutant was 20 times more sensitive to mercury than wild-type AqpZ and four times more sensitive than T183C. The X-ray structure of L170C shows four mercury atoms at, or near, the pore site defined in the T183C structure and no structural change upon mercury binding. Thus, we elucidate a steric inhibition mechanism for this important class of channels by mercury.
水通道蛋白家族的通道是根据汞化合物对水运输的抑制作用来定义的。尽管汞化合物起着重要作用，但对于汞结合导致通道抑制所涉及的结构变化却知之甚少。为了阐明其机制，我们设计了一种水通道蛋白Z（AqpZ）的突变体T183C，它是根据水通道蛋白1（AQP1）已知的汞敏感位点构建的，并确定了未结合状态和汞阻断状态下的X射线晶体结构。两种结构的叠加显示，汞结合后没有构象重排。在阻断结构中，有两个汞位点，一个与半胱氨酸183结合并堵塞孔道，另一个也与同一个半胱氨酸结合，但位于间隙腔中。为了测试阻断机制，我们设计了另一种突变体L170C，以便在孔道位点产生更有效的汞阻断。在剂量反应抑制研究中，该突变体对汞的敏感性比野生型AqpZ高20倍，比T183C高4倍。L170C的X射线结构显示，在T183C结构定义的孔道位点或其附近有四个汞原子，汞结合后没有结构变化。因此，我们阐明了汞对这类重要通道的空间抑制机制。
