Mechanistic Insights into G-protein Activation via Phosphorylation Mediated Non-Canonical Pathway

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Activation of heterotrimeric G-proteins (G αβγ ) downstream to receptor tyrosine kinases (RTKs) is a well-established crosstalk between the signaling pathways mediated by G-protein coupled receptors (GPCRs) and RTKs. While GPCR serves as a guanine exchange factor (GEF) in the canonical activation of G α that facilitates the exchange of GDP for GTP, the mechanism through which RTK phosphorylations induce G α activation remains unclear. Recent experimental studies revealed that the epidermal growth factor receptor (EGFR), a well-known RTK, phosphorylates the helical domain tyrosine residues Y154 and Y155 and accelerates the GDP release from the G α i3, a subtype of G α -protein. Using well-tempered metadynamics and extensive unbiased molecular dynamics simulations, we captured the GDP release event and identified the intermediates between bound and unbound states through Markov state models. The additional negative charges introduced by phosphorylations rewired the inter-residue interactions and significantly weakened the salt bridges at the domain interface, contributing to the increased separation of the Ras-like and helical domains of G-protein. Furthermore, the unfolding of helix α F resulted in greater flexibility near the hinge region, facilitating a greater distance between domains in the phosphorylated G α i3. The release of GDP in the phosphorylated G-protein occurred at a faster rate compared to the unphosphorylated state, caused by increased fluctuations in conserved regions of P-loop, switch 1, and switch 2. Overall, this study provides atomistic insights into the activation of G-proteins induced by RTK phosphorylations and identifies the specific structural motifs involved in the process. The knowledge gained from the study could establish a foundation for targeting non-canonical signaling pathways and developing therapeutic strategies against the ailments associated with dysregulated G-protein signaling.

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