Structural Basis for Activation of Gαi by Non‐Receptor Guanine Nucleotide Exchange Modulators (GEMs)
Authors: Kalogriopoulos Nicholas Antonios , Rees Steven , Aznar Nicolas , Sun Nina , Chang Geoffrey , Ghosh Pradipta , Kufareva Irina 
Published: November 2023

Canonical signaling of heterotrimeric G proteins is initiated when ligand‐activated G protein‐coupled receptors (GPCRs) stimulate GDP‐to‐GTP nucleotide exchange in the a subunit with subsequent dissociation of the abg trimer. Structural basis of this activation has been elucidated as key steps in this process have been resolved at an atomic‐level through a string of major breakthroughs over the past two decades. Heterotrimeric G proteins can also be activated downstream of a variety of other receptors via members of a novel family of non‐receptor guanine nucleotide exchange modulators (GEMs). The preeminent member of the GEM family, GIV (a.k.a. Girdin), can activate Gαi downstream of several different types of receptors, including growth factor receptor tyrosine kinases (RTKs) and integrins. However, the structural basis for this activation has remained unsolved.

Here we present a 2.0 Å crystal structure of Gαi3 in complex with a GIV peptide that contains the prototype sequence motif necessary for activating Gαi. This structure reveals that GIV binds and triggers GDP release from Gαi in a manner that is distinct from GPCRs; while GPCRs bind the C‐terminal helix of Gai complexed with Gβγ and favor an internal structural rearrangement of the Ras domain that loosens GDP, GEMs accomplish the same by binding Gαi (either free monomers, or monomers competitively extracted from Go‐Loco‐bound or Gαi‐βγ trimers) within the hydrophobic cleft between the switch‐II and α3‐helix. Such binding is accompanied by a series of structural rearrangements pivotally involving Q204 in the switch‐II of Gαi. This residue is well‐known for its catalytic role in GTP hydrolysis. Through comparative analysis with previously solved structures, we found that in the presence of Gbg or GDIs, Q204 stabilizes switch‐I and, consequently, the overall high GDP affinity configuration of the nucleotide binding pocket. Our structure shows that GIV binding tugs Q204 upward/outward >11 Å from switch‐I, thus releasing switch‐I and allosterically destabilizing the bound GDP molecule. The observed structural rearrangements are propagated through the G protein loosening the inter‐domain packing which likely results in an overall ‘low‐affinity state’ for GDP and facilitates GDP release. The structure also provides a high‐resolution insight into how key phospho‐modifications at strategic locations turns GIV “on” or “off”. Because the GIV‐family GEMs have emerged as consequential signaling components in diverse pathophysiologic states, these atomic‐level insights will enable the application of structure‐based approaches to develop better strategies for selectively targeting this pathway.

Support or Funding Information

This work was supported by NIH grants CA100768, CA160911 and DK099226 (to P.G). P.G. was also supported by the Burroughs Wellcome Fund (CAMS award), the American Cancer Society (ACS‐IRG 70‐002) and by the UC San Diego Moores Cancer Center. I.K. was supported by NIH grants GM071872 and AI118985. N.A.K. was supported by the NCI/NIH (5T32CA067754‐17).