21 April 2020, Version 1
This content is a preprint and has not undergone peer review at the time of posting.


Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the glob-al conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. Here, we describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix aC. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix aC in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix aC can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells.


conformation-selective inhibitors
ATP-competitive inhibitors
SH3 domain
Global Conformations
DFG-OUT conformation
ATP-binding site interactions
Tyrosine Kinase
drug sensitization
Drug Resistant


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