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Download Targe(ng the plas(city of Abl and cSrc tyrosine kinases through
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Targe&ng the plas&city of Abl and cSrc tyrosine kinases through mutagenesis and atomically detailed large-‐scale molecular dynamics simula&ons Ludovico Sutto! Computational Biophysics Group! Structural Biology and Biocomputing Programme! 1st July 2010 III RES user’s conference -‐ Zaragoza Abl, cSRC members of a large family Protein Kinases are key regulators of cell func&on (signal transduc&on, cellular growth/development/death..) Abl – cSRC sequence iden&ty 46% Protein Kinase func&on ac&vated Protein Kinase + protein + ATP CATALIZE THE PHOSPHORILATION OF phosphorilated protein A PROTEIN SUBSTRATE • same reac&on -‐> same ac&ve conforma&on • family members differ in inac&ve conforma&on and ac&va&on mechanism • wrong ac&va&on may lead to cancer ADP Inac&ve states of ABL and cSRC featuring DFG flip Ima+nib binds to DFG-‐Asp out conformaCons -‐> potent anC-‐cancer drug Affinity for cSRC >2000x lower (from nM to µM) cSRC exists also in DFG-‐Asp out inac&ve conforma&on cSRC inacCve state ABL-‐like DFG-‐Asp out , Aloop closed Ima-nib binds to c-‐SRC with the same orienta&on and interac&ons as in Abl. Nevertheless the affinity is much lower. Seeliger M.A. et al. (2007) Structure 15: 299-311 Single point muta&on in cSRC increases Ima&nib affinity Muta&on at the hydrophobic INTERFACE between the N-‐lobe and the C-‐lobe L317 WT/ STI L317I/ STI Computa&onal approach All-‐atoms in explicit solvent • ~30k atoms • precise conforma&onal ensembles • force field validated by many experiments gecng more and more reliable • computa&onally expensive Time scale problem for proteins atom vibra&ons large local conforma&onal equilibra&on transi&ons folding &me 10-‐15 s 10-‐9 s 10-‐6 s 10-‐3 s Enhanced sampling methods Metadynamics(1) • evolving bias discourages revisi&ng conforma&ons already explored • reconstruct the FES • choice of CV cri&cal replica exchange methods to further increase the sampling : bias exchange(2) and parallel tempering(3) metadynamics (1) Laio, A., and M. Parrinello, Proc. Natl. Acad. Sci. USA. 2002, 99:12562–12566 (2) S. Piana and A. Laio, J. Phys. Chem. B 111, 4553, 2007 (3) G. Bussi et al., J. Am. Chem. Soc., 2006, 128 (41), pp 13435–13441 movie by G. Bussi Chosen CVs for cSRC flip Collec&ve variables for metadynamics • • • • • • • Distance Lys45 – Glu60 Distance Lys45 – Asp154 Distance Glu60 – Arg135 Distance Glu60 – Arg159 Distance Phe155 – Leu67 Distance β1 – A-‐loop Dihedral combina&on φ154 ψ154 ψ157 Comparing FES of cSRC mutant to WT Dihedral ψ155 WT mutant Distance Phe155 – Leu67 The mutant allows for larger DFG-‐Phe movements DFG flip pathways in cSrc WT Different DFG flipping path! mutant has a second route mutant Conclusions -‐ perspec&ves • Protein Kinases key players in cell • DFG plas&city central for Ima&nib binding • MD simula&ons essen&al to inves&gate the dynamics: enhanced sampling + good CVs + parallel compu&ng • cSRC mutant allows a different flip pathway • protona&on state of DFG-‐Asp may play role for flip in ABL (Shan et al. PNAS 2009) Acknowledgments G. Saladino I. Mereu J. Juraszek L. SuZo N. Besker M. D’Abramo F.L. Gervasio Computa-onal Biophysics Group A. CrisCani L. Scapozzo
