Modeling Structural and Functional Mechanisms of TRP Channels

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We have just started this project, so the progress is limited. We are collaborating with Sergei Sukharevs and Ching Kungs laboratories to develop structural models of the TRPy1 mechanosensitive channel from yeast. This project is a continuation of a very productive collaboration with these groups on modeling microbial mechanosensitive channels; i.e., we worked with them to develop models of the gating-mechanism of the large mechanosensitive channel MscL. The principal features of our MscL models have been validated by numerous groups, and are currently accepted. The project is also natural for us since we have been modeling the structures and functional mechanisms of homologous voltage-gated channels for over two decades. We have developed tentative alignments of the TRPy1 (target protein) sequences with those of potassium channels that have known crystal structures. These crystal structures are used as template structures for homology modeling. Voltage-gated channels are composed of two transmembrane domains; a centrally located pore-forming domain formed by S5-P-S6 segments and a more peripheral voltage-sensing domain formed by S1-S4 segments. Preliminary models have been constructed of the pore-forming domains (S5-P-S6 segments) of TRPy1 in open and closed conformations, and we should soon have models of the voltage-sensing domain (S1-S4) as well. However, the target and template sequences are so distant that the accuracy of the alignment is questionable, and some portions, including the ion selective region, is so different that K+ channel structures cannot be used as a template. We are using alternative methods that have proven successful in the past to model these regions. We plan to use both computational and experimental approaches to test and improve the preliminary models. The computational methods will involve extensive molecular dynamic simulations of the model structures embedded in a lipid membrane with water and ions on each side of the membrane and in the central pore. A new computational method developed by Andriy Anishkin of the Sukharev lab will be used. This method uses soft symmetry restraints that can be turned on and off during the simulations. The simulations will be performed initially without symmetry restraints to better allow the structure to move toward lower energy states, and then the symmetry restraints will be used to restore the four-fold symmetry of the model. These simulations will be performed with a number of alternative starting models based primarily on different alignments to try to identify the better models. The Kung lab has already performed numerous mutagenesis experiments on TRPy1, and has identified several mutations that dramatically alter gating properties of the channels. Our preliminary models will be examined to determine whether they are consistent with these data. Next, additional mutagenesis experiments will be performed to test various models. These experiments will likely include cross-linking experiments to determine whether residue pairs that are predicted to interact in specific states do in fact interact in those states. They will also involve residues that are predicted by the models to be crucial in determining the ion selectivity of the channel. In developing structural models of proteins, the most difficult part is typically developing the first model of a member of a family. After that, other related members that normally have similar backbone structures can be developed more easily using relatively standard homology modeling methods. We have selected TRPy1 to model first because our experimental collaborators can test the preliminary models. Once satisfactory models of the TRPy1 channel have been developed and tested, we will extend the models to human TRP channels that are of more interest biomedically.