Structure and functional mechanisms of molecular chaperones and protein kinases

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PROJECT SUMMARY Our lab works on two main directions: first, the determination of the structural and dynamic basis for the function and assembly of large protein machineries; and second, the determination of the role of internal protein dynamics in regulating protein activity and allosteric interactions. We propose to use NMR spectroscopy, together with other biochemical and biophysical techniques, to determine at the atomic resolution the mechanisms underpinning the function of two important protein families: molecular chaperones and protein kinases. Molecular chaperones are central to maintaining a functional proteome in the cell by rescuing non- native proteins from aggregation and misfolding and assisting with their folding. Our lab reported the first ever high resolution structures of molecular chaperones in complex with unfolded proteins. We will determine the structures of important chaperones such as the Hsp40, Hsp70 and Hsp90 in complex with client proteins. We wish to address how different chaperones engage non-native proteins and how distinct chaperone architectures may alter activity. Src and BRAF hold a prominent place among the over 500 protein kinases encoded by the human genome. Src is the archetypical kinase of the ~30 cytoplasmic tyrosine kinases, half of which (Src, Frk, Abl, Tec and Csk families) share the same core domain architecture consisting of an SH3, SH2 and kinase domain. As such, Src has served as the prototype for understanding how allosteric interactions regulate the activity of kinases. BRAF is a member of RAF kinases and is a part of the ERK signaling pathway, one of the most important and best studied signaling pathways in human cancers. The BRAF gene is mutated in approximately 8% of human tumors with substitutions in the A-loop, the P-loop and the DFG motif reported to give rise to a hyperactive kinase. We will use NMR spectroscopy to characterize transiently populated conformational states, with the goal of uncovering novel regulatory mechanisms in the human kinome.