Extended Data Fig. 10: Proposed molecular mechanism of P-Rex1 synergistic activation by Gβγ and PI(3,4,5)P_3.

a. Autoinhibited P-Rex1 may transiently interact with the inner leaflet of the plasma membrane via DEP1, DEP2, PH or the IP4P positively charged surfaces. b. Collectively, binding of Gβγ and DEP1, DEP2 and IP4P requires the counter-rotation of the N-terminal module (DH-PH-DEP1) relative to the C-terminal module (DEP2-PDZ1/2-IP4P:Gβγ). Movement of the PH domain toward the membrane is essential to unlock the autoinhibited DH domain. c. Counter-rotation of N- and C-modules disengages the 4HB and PH interaction, freeing the PH domain to bind PI(3,4,5)P₃. PI(3,4,5)P₃ binding causes the DH domain latch to unlock, thereby releasing the DH domain to bind Rac1. Autoinhibited P-Rex1 is modelled by the full-length cryo-EM reconstruction and AlphaFold²¹ (4HB regions). The cryo-EM reconstruction of Gβγ:P-Rex1 DEP2-PDZ1/2-IP4P¹⁵ allows placement of Gβγ:DEP2-PDZ1/2-IP4P against the bilayer (revealing offset planes defined by N- and C-modules). The crystal structure of PH: PI(3,4,5)P₃¹⁴, as well as functional data of DEP1/2²² membrane binding regions, guided the placement of the AlphaFold model of PH-DEP²¹ (kinked V-conformation) against the membrane. Lastly, superposition of the crystal structure of DH-PH:Rac1¹³ onto the membrane-anchored PH domain provides a model of the full active state. The Rac1 lipidation site provides additional short-range distance constraints on the placement of DH-PH:Rac1.