An Allosteric Region of Src Tyrosine Kinase Allows for Stabilization of Its Active-Like Conformation

Abstract

Protein kinases play an essential role as signaling enzymes and their sensitive action in the needs of the cell requires their tight regulation. Their active conformations are exquisitely regulated and their populations controlled by various post-translational modifications. The inactive conformation of Src tyrosine kinase is characterized by its non-phosphorylated activation loop and the rolled-out αC helix in the N-lobe. The inactive conformation has the regulatory kinase spine disassembled as the RS3 residue from the αC helix is displaced. Our umbrella sampling methods to map the energy landscape of these conformations has allowed for defining four states; the inactive, active conformations and two intermediate states. In the present study we extend our umbrella sampling technique to understand the activation allostery in Src tyrosine kinase in three different mutants. The first mutant is the L297F that occupies an allosteric region in the N-terminal of the αC helix and allows for activation of Src tyrosine kinase analogous to RAFs. The second mutant is the V281F mutation in the catalytic spine that allows for the closing of the two kinase lobes in the absence of nucleotide. A double mutant of Src tyrosine kinase V281F/L297F allows for stabilization of a unique catalytically dead kinase conformation in the active-like state. This unique conformation allows for understanding of kinase switching function independent of its catalytic role. A third M314V mutation in the regulatory spine allows us to explore the maneuvering of hydrophobicity in the Src tyrosine kinase core. These studies provide a fresh perspective to understanding conformational switching in Src family of tyrosine kinases and the dynamic allostery that underlies this molecular communication. Our community map analysis of residue networks provides an additional layer of understanding to these observations.