Huang Yiping, Nokhrin Sergiy, Hassanzadeh-Ghassabeh Gholamreza, Yu Corey H, Yang Haojun, Barry Amanda N, Tonelli Marco, Markley John L, Muyldermans Serge, Dmitriev Oleg Y, Lutsenko Svetlana
From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
the Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.
J Biol Chem. 2014 Nov 21;289(47):32682-93. doi: 10.1074/jbc.M114.580845. Epub 2014 Sep 24.
The biologically and clinically important membrane transporters are challenging proteins to study because of their low level of expression, multidomain structure, and complex molecular dynamics that underlies their activity. ATP7B is a copper transporter that traffics between the intracellular compartments in response to copper elevation. The N-terminal domain of ATP7B (N-ATP7B) is involved in binding copper, but the role of this domain in trafficking is controversial. To clarify the role of N-ATP7B, we generated nanobodies that interact with ATP7B in vitro and in cells. In solution NMR studies, nanobodies revealed the spatial organization of N-ATP7B by detecting transient functionally relevant interactions between metal-binding domains 1-3. Modulation of these interactions by nanobodies in cells enhanced relocalization of the endogenous ATP7B toward the plasma membrane linking molecular and cellular dynamics of the transporter. Stimulation of ATP7B trafficking by nanobodies in the absence of elevated copper provides direct evidence for the important role of N-ATP7B structural dynamics in regulation of ATP7B localization in a cell.
由于其低表达水平、多结构域结构以及作为其活性基础的复杂分子动力学，生物学和临床上重要的膜转运蛋白是具有挑战性的研究对象。ATP7B是一种铜转运蛋白，可响应铜浓度升高在细胞内区室之间运输。ATP7B的N端结构域（N-ATP7B）参与铜的结合，但该结构域在运输中的作用存在争议。为了阐明N-ATP7B的作用，我们制备了在体外和细胞中与ATP7B相互作用的纳米抗体。在溶液核磁共振研究中，纳米抗体通过检测金属结合结构域1-3之间短暂的功能相关相互作用，揭示了N-ATP7B的空间组织。纳米抗体在细胞中对这些相互作用的调节增强了内源性ATP7B向质膜的重新定位，将转运蛋白的分子和细胞动力学联系起来。在没有铜浓度升高的情况下，纳米抗体对ATP7B运输的刺激为N-ATP7B结构动力学在调节细胞中ATP7B定位的重要作用提供了直接证据。
