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| Richard Dixon, University of North Carolina at Chapel Hill |
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Detection of Intermediate States of Cell Adhesion Proteins Using Discrete Molecular Dynamics (DMD) Simulations Constrained By Nuclear Magnetic Resonance (NMR) Hydrogen Exchange (HX) Data
Richard D.S. Dixon, Yiwen Chen, Feng Deng, Sagar D. Khare, Kirk C. Prutzman, Sean M. Palmer, Michael D. Schaller, Nikolay V. Dokholyan, and Sharon L. Campbell, Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA
We are interested in characterizing conformational dynamic properties of two important cell adhesion proteins, focal adhesion kinase (FAK) and vinculin, which appear to play important roles in mediating their respective tumor promoting and tumor suppressor properties. We have combined NMR spectroscopy with computational approaches to investigate conformational dynamic properties of these two systems. Specifically, DMD simulations constrained by NMR-derived protection factors have been used to detect intermediate states associated with the focal adhesion targeting (FAT) domain of FAK [1] and the tail domain (Vt) of vinculin. Our studies have revealed an intermediate state of the FAT domain in which helix 1 partitions from the native four-helix bundle. These results are consistent with an intermediate state of FAT that promotes phosphorylation within helix 1 [1], prevents paxillin binding [1] and facilitates formation of a domain swapped dimer [2]. Thus, we and others have proposed that these conformationally dynamic sub-states of the FAT domain of FAK regulate the sub-cellular localization of FAK, ligand binding and consequently growth factor and integrin-mediated signaling. We are currently testing this hypothesis through the use of mutants that alter the conformational dynamic properties of FAT in conjunction with cell biology, biochemical and biophysical approaches.
We have also initiated DMD/HX studies on the tail domain of vinculin (Vt), which, upon activation, dissociates from the head domain of vinculin and interacts with multiple binding partners, including paxillin, acidic phospholipids and F-actin, to promote integrin-mediated actin assembly. Vt is a 5-helix bundle domain that shows structural similarity to the FAT domain. Moreover, distinct conformations of Vt have been proposed to modulate ligand binding. Our simulations on Vt present us with the reverse case to that of the FAT domain of FAK; phosphorylation sites and ligand-binding interfaces of the tail domain of vinculin remain to be characterized so future studies will reveal the biological role of the intermediate states that our simulations have produced. For both the FAT domain and Vt, the integration of experimental and computational methods has provided insights into the dynamic features of these molecules that could not be obtained solely from conventional experiments.
[1] Dixon, R.D.S, Chen, Y., Feng, D., Khare, S.D., Prutzman,K.C., Schaller, M.D., Campbell, S.L., Dokholyan, N.V. (2004), Structure, 12(12), 2161-2171.
[2] Feng, D., Prutzman, K.C., Campbell, S. L., Dokholyan, N.V. submitted.
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