Mechanism Of Calcium Binding To TMEM16A Channel Via Electrostatic Interactions

Calcium (Ca2+) is one of the most common second messenger and contributes to allaspects of the cell’s function. Calcium and calcium-binding protein (CaBPs)，whichcombined by electrostatic interactions, regulate cell physiological activity. Research for thebinding mechanism of calcium is one of the hot topics in biology, chemistry and physics.As molecular identity of calcium-activated chloride channels (CaCCs), transmembraneprotein16A (TMEM16A) is dual regulated by calcium and voltage. Study on the Ca2+binding mechanism is helpful for understanding the calcium dependence of CaCCs.However, there is neither crystal structure of TMEM16A nor any obvious EF hand or C2domain like Ca2+-binding sites in its primary sequence. Because TMEM16A has lowermolecular identity with other proteins, it is impossible to build the steric structure of it byhomology modeling. Moreover, a straightforward approach to identify binding site ofTMEM16A would be alanine scan which turned out to be a roundabout andtime-consuming process for such a big protein. All of the obstacles make the topic apending scientific problem.In this study, we addressed two issues,1) Validate if we can identify Ca2+bindingsites through homology modeling and MD simulations.2) Probe the Ca2+binding sites inTMEM16A.1. Here we performed a computational approach which combined the fragmenthomology modeling with MD simulation aimed at identifying the Ca2+-binding site inCalcium binding proteins without crystal structures. Our data indicated that the predictionaccuracy in CaBPs with known crystal structure was70%～80%, and the data of CaBPswith unknown crystal structure, BK channel and TMEM16A, are consistent with theexperimental results. Our work will shed light on prediction of the binding site in proteinswhich have neither crystal structure nor typical calcium binding site.2. We combined theoretical prediction with site direct mutation andelectrophysiological experiments to analyze the pattern and site of Ca2+binding inTMEM16A. The data shows that the motif consisted of D439, E444, and E447in the fistintracellular loop acts as a Ca2+binding site. Especially, both the electro neutral mutateE447A and the flexile mutate E447Y turned out to cause a dramatic decrease of Ca2+ apparent affinity of TMEM16A.3. To evaluate the contribution of D439, E444, and E447on binding Ca2+, wecalculated PMF, the data shows that two carbonyl oxygen atoms of the E447binding withCa2+by electrostatic interaction, therefore, mutation of E447leads to greater effect on theapparent affinity than the other residues.