Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

Abstract

P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.

Structural and dynamic changes in P-Rex1 upon activation by PIP3and inhibition by IP4

“…This apparent contradiction can now be explained by the fact that in DH/PH-DEP1 and larger fragments, the DH/PH module can jack-knife and position the PH domain in a manner that blocks GTPase binding. This model of regulation was corroborated by recent structural studies of P-Rex1 without IP 4 (Chang et al, 2022). The isolated DEP1 domain was also shown to play an autoinhibitory role based on the relatively low activity of DH/PH-DEP1 relative to DH/PH (Ravala et al, 2020).…”

“…While conducting these studies, the structure of human P-Rex1 in the absence of IP 4 was reported (PDB entry 7SYF) (Chang et al, 2022), allowing a comparison between the IP 4 and IP 4 -free states of autoinhibited P-Rex1. Overall, the domain organization is very similar, but there is an ∼3° rotation of the Gβγ-binding core in the IP 4 complex relative to the PH–4HB interface such that DEP1 and DEP2 move closer together.…”

“…While conducting these studies, the structure of human P-Rex1 in the absence of IP 4 was reported (PDB entry 7SYF) (Chang et al, 2022), allowing a comparison between the IP 4 and IP 4 -free states of autoinhibited P-Rex1 (Sup. Fig.…”

In vitro reconstitution of small GTPase regulation

“…Intra‐ and intermolecular interactions of the regulatory domains control the catalytic activity and define the intracellular location of the GEF complex. Several layers of autoregulation can efficiently control where and when a small GTPase is activated in the cell [52,96]. The hierarchy of these interactions and possible cooperative or combinatorial effects often still need to be discovered.…”

Small-molecule inhibitors of P-Rex guanine-nucleotide exchange factors

“…Furthermore, a cryo-EM structure of a large portion of P-Rex1 from the second DEP domain to the C-terminus confirmed extensive autoinhibitory intramolecular interactions and their relief through allosteric changes brought about by Gβγ binding [ 39 ]. Recently, a crystal structure including the domains from the DH to the DEP1 domain and a cryo-EM structure of the full-length P-Rex1 protein have been solved by the Ellisdon lab, revealing a two-layered mechanism to relieve P-Rex1 autoinhibiton, whereby P-Rex1 binding to PIP 3 and Gβγ causes first a rotation between the N- and C-terminal halves of the GEF and then an opening of the DH domain that enables Rac binding and catalysis [ 40 ].…”