PXD034327-1

PXD034327 is an original dataset announced via ProteomeXchange.

Title	Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism
Description	Rho GTPases regulate actin dynamics and cell motility, and their hyperactivation drives cancer metastasis1. P-Rex (PI(3,4,5)P3-dependent Rac Exchanger) guanine nucleotide exchange factors (GEFs) are potent on-switches for Rho GTPase signalling and are frequently dysregulated in metastatic cancer2. P-Rex GEFs are autoinhibited under basal conditions and activated synergistically by binding to Gβγ and PI(3,4,5)P33. However, the molecular basis for P-Rex autoinhibition remains unknown. Here we utilise X-ray crystallography, cryo-EM and cross-linking mass spectrometry to determine the autoinhibited P-Rex1 structure. P-Rex1 forms a characteristic bipartite structure with its seven domains split into distinct N- and C-terminal modules. The two modules are connected by a previously unrecognised C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the catalytic surface of the Dbl homology (DH) domain is occluded by the compact arrangement of the PH and DEP1 domains. Structural analysis of the DH-PH-DEP1 array reveals that a remarkable conformational transition, centred on a 126° opening of a hinge helix reminiscent of calmodulin dynamics, leads to the release of DH domain autoinhibition. Furthermore, given the off-axis position of the Gβγ and PI(3,4,5)P3 binding sites, our data suggest that concurrent Gβγ and PI(3,4,5)P3 requires counter-rotation of the two halves of P-Rex1 by 90°, leading to the uncoupling of the PH domain from the four-helic¬¬al bundle, and release of the autoinhibited DH domain to drive Rho GTPase signalling.
HostingRepository	PRIDE
AnnounceDate	2022-08-12
AnnouncementXML	Submission_2022-08-12_06:22:15.363.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ralf Schittenhelm
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Q Exactive HF

Revision	Datetime	Status	ChangeLog Entry
0	2022-06-06 00:56:29	ID requested
⏵ 1	2022-08-12 06:22:15	announced
2	2023-11-14 08:57:08	announced	2023-11-14: Updated project metadata.
3	2024-10-22 05:39:07	announced	2024-10-22: Updated project metadata.

10.1038/S41594-022-00804-9;

submitter keyword: P-Rex1, Rac1, Cryo-EM, cancer, metastasis

Andrew Ellisdon
contact affiliation	Biomedicine Discovery Institute, Monash University, Clayton 3800, Victoria, Australia
contact email	Andrew.ellisdon@monash.edu
lab head
Ralf Schittenhelm
contact affiliation	Monash University
contact email	ralf.schittenhelm@monash.edu
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD034327
     1. Label: PRIDE project
     2. Name: Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism