An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

Cuesta, N.; Contreras, J. M.; Sanchez-Waldermer, J.; Soriano-Maldonado, P.; Martin-Hurtado, A.; Munoz, I. M.; Llimargas, M.; Munoz, J.; Plaza-Menacho, I.

doi:10.1101/2022.10.16.512342

bioRxiv · 10.1101/2022.10.16.512342

An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

Cuesta, N.·Contreras, J. M.·Sanchez-Waldermer, J.·Soriano-Maldonado, P.·Martin-Hurtado, A.·Munoz, I. M.·Llimargas, M.·Munoz, J.·Plaza-Menacho, I.

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Abstract

Auto-phosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlaying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src auto-phosphorylation site with slow time-resolution kinetics and strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes fast kinetics and a cis-to-trans phosphorylation-switch that controls c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular phosphorylation in which a c-terminal palindromic phospho-motif flanking Tyr 530 on the susbtrate molecule engages the P-loop of the active kinase for ready entry prior catalysis. Perturbation of the phospho-motif accounts for c-Src disfunction as indicated by viral and a colorectal cancer (CRC) associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 and global auto-phosphorylation, and this detrimental effect is caused by the susbtrate molecule inhibiting allosterically the active kinase. Our work reveals a bi-directional crosstalk between the activation and c-terminal segments that controls the allosteric interplay between susbtrate and enzyme acting kinases during auto-phosphorylation HighlightsO_LIA bi-directional phospho-switch connecting the activation and c-terminal segments controls c-Src function C_LIO_LIActivation-loop Tyr 419 is required for c-terminal Tyr 530 auto-phosphorylation, enzyme specificity and non-catalytic function as a substrate C_LIO_LIBiochemical and structural visualization of c-Src Tyr 530 intermolecular auto-phosphorylation C_LIO_LIA double-phosphorylated c-Src on both the activation and c-terminal segments is a fully active protein C_LIO_LICancer associated c-terminal deleted variants inhibit allosterically c-Src activity by a dominant negative effect C_LI

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Cuesta, N., Contreras, J. M., Sanchez-Waldermer, J., Soriano-Maldonado, P., Martin-Hurtado, A., Munoz, I. M., Llimargas, M., Munoz, J., Plaza-Menacho, I.. 2022-10-16. An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function. https://doi.org/10.1101/2022.10.16.512342

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