School of Life Sciences, University of Technology Sydney, Sydney, New South Wales 2007, Australia.
Australian Nuclear Science and Technology Organisation (ANSTO), Lucas Height, Australian Centre for Neutron Scattering, New South Wales 2234, Australia.
Biochim Biophys Acta Gen Subj. 2019 Aug;1863(8):1243-1253. doi: 10.1016/j.bbagen.2019.04.020. Epub 2019 May 8.
Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol.
We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol.
Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane.
In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels.
Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting activity and a novel regulatory role for cholesterol in such a process.
固醇已被报道可调节几种膜蛋白的构象和功能。氯离子细胞内离子通道（CLIC）家族的蛋白质就是这样的一个群体。CLIC 蛋白家族在脊椎动物中由六个进化上保守的蛋白成员组成。这些蛋白质既可以作为单体可溶性蛋白存在，也可以作为膜结合蛋白存在。迄今为止，其膜结合形式的结构仍然未知。除了几项研究表明细胞氧化还原环境和 pH 值有助于 CLIC1 插入膜中外，我们还证明 CLIC1 的自发膜插入受膜胆固醇调节。
我们进行了 Langmuir 膜、阻抗谱和分子对接模拟，以研究该 GXXXG 基序在 CLIC1 与胆固醇相互作用中的作用。
与 CLIC1-野生型蛋白不同，形成 GXXXG 基序一部分的 G18A 和 G22A 突变体与天然蛋白相比，表现出较慢的初始动力学和较低的离子通道活性。这种差异可归因于突变蛋白与膜的相互作用和插入速率显著降低，或者一旦进入膜中，突变蛋白的最终膜构象的形成速度较慢。
在这项研究中，我们的发现揭示了 CLIC1 中存在 GXXXG 基序，该基序可能作为胆固醇结合域，促进蛋白质的膜相互作用和插入。此外，我们能够假设一个模型，即 CLIC1 可以自主插入膜中形成功能性离子通道。
CLIC 家族的蛋白质成员表现出异常的结构和双重功能特性——作为离子通道和酶。阐明 CLIC 蛋白如何与膜相互作用，从而使它们在可溶性和膜形式之间切换，将为 moonlighting 活性的机制以及胆固醇在该过程中的新型调节作用提供关键信息。
