Structural and dynamic changes in P-Rex1 upon activation by PIP3 and inhibition by IP4

Sandeep K. Ravala
Sendi Rafael Adame-Garcia
Sheng Li
Chun-Liang Chen
Michael A. Cianfrocco
J. Silvio Gutkind
Jennifer N. Cash author has email address
John J. G. Tesmer author has email address

Departments of Biological Sciences and of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907, United States
Department of Pharmacology and Moores Cancer Center, University of California, San Diego, San Diego, CA 92093, USA
Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States
Department of Molecular and Cellular Biology, University of California-Davis, Davis, CA, 95616, USA

https://doi.org/10.7554/eLife.92822.2

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Figures and data

IP₄ binding causes dynamic changes in multiple domains of P-Rex1 and inhibits PIP₃-induced activation.
A) Difference HDX-MS data plotted onto the domain layout of P-Rex1. Blue regions indicate less deuterium uptake upon IP₄ binding. Graphs show the exchange over time for select regions in the P-Rex1 (B) PH domain and (C) an IP4P region that was disordered in the P-Rex1–Gβγ structure. Shown is the average of two experiments with error bars representing the mean ± standard deviation. D) In vitro GEF activity of P-Rex1 evaluated on liposomes containing 2.5 μM PIP₃ in the presence of varying IP₄ concentrations (0-100 μM). Data were fit to exponentials to get rate constants by constraining the span to be shared. The resulting rates for each experiment were normalized by averaging two PIP₃ data points and two PC/PS data points to represent the top and bottom of the binding curve. The resulting normalized rates (min⁻¹) were fit with a one-phase binding curve wherein the top and bottom were constrained to 1 and 0, respectively, and the Hill coefficient fixed at −1. The resulting IC₅₀ was 1.4 µM with a confidence interval of 0.81 to 2.3. Data represent 4-5 independent experiments. Error bars represent the mean ± S.D.

Structure of the P-Rex1·IP₄ complex in an autoinhibited conformation.
A) Cryo-EM reconstruction with atomic model superimposed. The kink between the DH and PH domains and the GTPase binding site are labeled. B) Atomic model without the cryo-EM map. C) The PH–4HB interface primarily involves the β1/β2 and β5/β6 loops of the PH domain, which were previously shown to be involved in protein-protein interactions in crystal structures (Cash et al., 2016), and the 4HB₁ and 4HB₂ helices of the 4HB domain. Flexible loops, including the basic β3/β4 loop of the PH domain involved in membrane binding (Cash et al., 2016), are shown as dashed lines. We speculate that this loop could interact with phosphorylated residues in the adjacent 4HB unstructured loop. D) Side chains in the PH–4HB interface. The 3-, 4-, and 5-position phosphates of bound IP₄ are labeled. Note that PIP₃ could not bind to the PH domain in this state due to steric blockade by the 4HB domain. The area of focus in (C) and (D) is circled in transparent grey in (B).

Cryo-EM data collection, refinement, and validation statistics

Cryo-EM data collection, refinement, and validation statistics

Mutations at the DH–DEP1 interface alter stability, conformation, and activity of DH/PH-DEP1.
A) Side chains that contribute to the hydrophobic interface formed between the DH and DEP1 domains. B) Electrostatic interactions contributing the DH–DEP1 interface. The dotted line indicates a disordered region on the DH domain containing positively charged residues that may interact with Glu456. The A170K mutant is expected to form a salt bridge with Glu411 and strengthen the interface. C) Fluorescence based in vitro GEF activity assay on soluble Cdc42 with variants of the purified DH/PH-DEP1 fragment. GEF activity in this experiment was fit to a one phase exponential decay normalized to that of DH/PH-DEP1 (WT). ****, P<0.0001. D) Representative Thermofluor analyses showing that mutations that disrupt the DH–DEP1 interface also destabilize the protein, as evidenced by decreased T_m values for each variant (see Table 2). Data are normalized from 0-100% representing lowest and highest fluorescence values. Note that A170K, which inhibits activity in panel C, increases stability. E, F) EOM analysis of SAXS data collected from mutations disrupting the DH–DEP1 interface indicate that these variants exhibit more extended conformations (see Table 3). EOM analyses provide the R_g and D_max distributions derived from selected ensembles. The gray curves correspond to the R_g and D_max distributions for the pool of structures used for each analysis.

Thermofluor measurements of DH/PH-DEP1 variants

SAXS parameters for DH/PH-DEP1 variants

HDX-MS and cryo-EM data support that IP₄ stabilizes a closed conformation of P-Rex1.
A) Difference HDX-MS data plotted onto the structure of the P-Rex1 bound to IP₄. Blue regions indicate more protection upon IP₄ binding whereas red regions indicate less. See also Supplemental Data 1. B) Map representing IP₄ bound in the PIP₃-binding site of the PH domain. The 3-, 4-, and 5-phosphates of IP₄ are reasonably well-ordered.

Disruption of the DH–DEP1 and PH–4HB interfaces leads to increased P-Rex1 activity in cells.
A) SRE luciferase-gene reporter assays. Mutations were cloned into full-length P-Rex1 in the pCEFL-HA-HaloTag vector, and these constructs, along with luciferase reporter genes, were co-transfected into HEK293T cells. Results depicted here are representative of three independent experiments, and error bars represent S.D. Non-transfected control (C) and empty vector transfected control (Halo) are shown. B) Mutations which led to enhanced P-Rex1 activity in luciferase reporter assays were evaluated for their effect on chemotaxis of HeLa cells with endogenous P-Rex1 knocked out (HeLa P-Rex1 KO; see Supplemental Figure 7). P-Rex1 constructs were transfected into HeLa P-Rex1 KO cells, and cell migration was evaluated in a trans-well migration assay upon stimulation with CXCL12 (50 ng/ml) or EGF (50 ng/ml). Data is presented as mean ± S.D. Significance (brackets) was determined using multiple comparison ANOVA followed by Šidák statistic test.

HDX-MS supports that P-Rex1 undergoes long range conformational changes when binding PIP₃-containing liposomes.
A) HDX-MS of P-Rex1 in the presence of PIP₃-containing liposomes. A model of P-Rex1 in an open conformation bound to a membrane containing PIP₃ was created and is shown colored according to difference HDX-MS data plotted onto the coordinates. HDX-MS data were collected in the presence of liposomes containing PIP₃ and compared to data collected on P-Rex1 alone. Blue and red regions indicate less and more protection, respectively, upon PIP₃-containing liposome binding. These changes occur specifically in the presence of PIP₃ (see Supplemental Figure 8). The black line at the top represents a membrane surface and the dashed lines represent covalent lipid modifications. Using available structural information, Gβγ and Rac1 were docked into this model (although neither were present in this HDX-MS experiment). B) Cartoon schematic of our model of the steps involved in the activation of P-Rex1.

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