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

  1. Sandeep K. Ravala
  2. Sendi Rafael Adame-Garcia
  3. Sheng Li
  4. Chun-Liang Chen
  5. Michael A. Cianfrocco
  6. J. Silvio Gutkind
  7. Jennifer N. Cash
  8. John J. G. Tesmer

  • Curated by eLife

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    This important study contributes insights into the regulatory mechanisms of a protein governing cell migration at the membrane. The integration of approaches revealing protein structure and dynamics provides convincing data for a model of regulation and suggests a new allosteric role for a solubilized phospholipid headgroup. The work will be interesting to researchers focusing on signaling mechanisms, cell motility, and cancer metathesis.

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Abstract

PIP 3 -dependent Rac exchanger 1 (P-Rex1) is abundantly expressed in neutrophils and plays central roles in chemotaxis and cancer metastasis by serving as a guanine-nucleotide exchange factor (GEF) for Rac. The enzyme is synergistically activated by PIP 3 and the heterotrimeric Gβγ subunits, but mechanistic details remain poorly understood. While investigating the regulation of P-Rex1 by PIP 3 , we discovered that Ins(1,3,4,5)P 4 (IP 4 ) inhibits P-Rex1 activity and induces large decreases in backbone dynamics in diverse regions of the protein. Cryo-electron microscopy analysis of the P-Rex1·IP 4 complex revealed a conformation wherein the pleckstrin homology (PH) domain occludes the active site of the Dbl homology (DH) domain. This configuration is stabilized by interactions between the first DEP domain (DEP1) and the DH domain and between the PH domain and a 4-helix bundle (4HB) subdomain that extends from the C-terminal domain of P-Rex1. Disruption of the DH–DEP1 interface in a DH/PH-DEP1 fragment enhanced activity and led to a more extended conformation in solution, whereas mutations that constrain the occluded conformation led to decreased GEF activity. Variants of full-length P-Rex1 in which the DH–DEP1 and PH–4HB interfaces were disturbed exhibited enhanced activity during chemokine-induced cell migration, confirming that the observed structure represents the autoinhibited state in living cells. Interactions with PIP 3 -containing liposomes led to disruption of these interfaces and increased dynamics protein-wide. Our results further suggest that inositol phosphates such as IP 4 help to inhibit basal P-Rex1 activity in neutrophils, similar to their inhibitory effects on phosphatidylinositol-3-kinase.

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  1. Version published to 10.7554/elife.92822.2 on eLife
  2. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    We thank the reviewers for their thorough review of and overall positive comments on our manuscript. We have revised the manuscript to address most of the concerns raised. Below is a point-by-point response to the reviewers’ comments outlining these changes.

    The novelty of the study is compromised due to the recently published structure of unliganded PRex1 (Chang et al. 2022). The unliganded and IP4-bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper, a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta/gamma is presented.

    This comparison has been added as Supplemental Figure 5. Although similar models of activation are presented in our manuscript and in that of Chang et al. 2022, our model is extended to incorporate inhibition by IP4 and other aspects of regulation not previously incorporated, shown in both schematic form (Figure 6B) and including supporting data (Figure 6A). We also point out that in the work by Chang et al. they used domain insertions to stabilize the structure, and here we present the native protein structure. It turns out that they look similar, but our work reduces concerns over possible engineering artifacts. Finally, our model is further informed by HDX-MS measurements of the enzyme bound to PIP3 in liposomes (Figure 6A and Supplemental figure 8), which reveal the regions of the protein subject to higher dynamics and are consistent with a more fully extended conformation.

    The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

    We feel that both occur. IP4 and PIP3 bind to the same site of the PH domain, thus they must be competitive at the very least. We also show that IP4 stabilizes the autoinhibited conformation (based on both our cryo-EM and HDX-MS data). Because PIP3 does not activate either DH/PH or DH/PH-DEP1 (nor does IP4 inhibit, see Sup. Fig. 1), it is not possible for us to tell with this suite of experiments how much the inhibition is due to competition versus stabilization of the autoinhibited conformation.

    It is difficult to judge the error in the HDX experiments presented in Sup. data 1 and 2. In the method section, it is stated that the results represent the average from two samples. How is the SD error calculated in Fig.1B-C?

    To clarify, the following passages have been revised:

    Figure 1 legend – “Graphs show the exchange over time for select regions in the P-Rex1 (B) PH domain and (C) a IP4P region that was disordered in the P-Rex1–Gbg structure. Shown is the average of two experiments with error bars representing the mean ± standard deviation.” Methods section – “Each sample was analyzed twice by HDX-MS, and the data shown in graphs represent the average of these experiments. For each peptide, the average of all five time points was calculated and used to plot the difference data onto the coordinates.”

    As mentioned, from the explanations in the manuscript it is difficult to judge the differences between the unliganded and the IP4 bound structure. A superposition, pointing to the main differences, would help. Are there any additional interactions observed that could explain a more stable autoinhibitory conformation?

    Added as Supplemental Figure 5. Although there are global shifts in some of the domains, the overall structures are similar to one another. Due to the moderate resolution of both structures (~4.2 Å), accurate placement of sidechains is difficult, in some places more than others. Because of this, we cannot pinpoint many specific sidechain interactions with certainty. There are no obvious interactions observed in our IP4 bound structure compared to that of 7SYF that would explain a more stable autoinhibited conformation, and thus the evidence comes primarily from the HDX-MS data.

    The cellular significance of IP4 regulation is not clear. Finding a way to manipulate intracellular IP4 levels and showing that this affects P-Rex1 cellular activity would greatly increase the significance of this finding.

    We agree that this would be an informative experiment, but not one that we currently have the means to perform.

    From the presented data it is not clear if inhibition by IP4 is due to competition with PIP3 or due to the proposed stabilization of P-Rex1 autoinhibition. Performing a study as shown in Fig.1D, but with the DH/PH construct could resolve this question.

    First, please see our response to the similar concern from Reviewer 1 above. It is not possible for us to test the DH/PH construct and assess if there is direct competition with PIP3. To emphasize this point (and to correct the error that we never made a call to Sup. Fig. 1C in the original manuscript), we added the following lines to the first paragraph of the Results.

    “Negatively charged liposomes (containing PC/PS), including those that also contain PIP3, unexpectedly inhibit the GEF activity of the DH/PH-DEP1 and DH/PH fragments (Sup. Fig. 1C). Because full-length P-Rex1 is not affected by PC/PS liposomes, it suggests this the observed inhibition represents a non-productive interaction of the DH/PH-DEP1 and DH/PH fragments with negatively charged surfaces in our assay. The lack of activation of DH/PH-DEP1 by PIP3 prevents us from testing whether IP4 can directly inhibit via direct competition with PIP3.”

    If I understand correctly, the data shown in Supplementary Data 1 and 2 are averages of 2 measurements, which makes it difficult to judge real signals from outliers. Perhaps, rather than showing the average, the results from the two experiments could be shown. Also, please explain how the SD error is calculated in Fig.1B-C if the data points indeed are averages of 2 measurements.

    We are sorry for the confusion. The data shown in Sup. Data 1 and 2 are not averages of two experiments. The Methods section has therefore been modified to read: “Each image in Supplemental Data 1 and 2 shows one experiment (rainbow plots) or a difference analysis from those experiments (red to blue plots). Only one of the two sets of experiments performed for each condition (+/- liposomes or +/- IP4) is shown here.” As described above, text has been added to clarify the SD error calculated in Fig. 1B and 1C.

    The authors claim that the data presented in Fig 4B suggests that the salt bridge formed by K207 and E251 is important for autoinhibition. If so, the authors should explain why the K207C mutant is not activated.

    Multiple reviewers had problems with this panel, and we now recognize that we misinterpreted the data, which did not help with this. Because this data is largely just supportive of our structure and SAXS data, Figure 4 was moved to the Supplement and this section of the results now reads:

    “Flexibility of the hinge in the a6-aN helix of the DH/PH module is important for autoinhibition.

    One of our initial goals in this project was to determine a high-resolution structure of the autoinhibited DH/PH-DEP1 core by X-ray crystallography. To this end, we started with the DH/PH-DEP1 A170K variant, which was more inhibited than wild-type but still dynamic, and then introduced S235C/M244C and K207C/E251C double mutants to completely constrain the hinge in the a6-aN helix via disulfide bond formation in a redox sensitive manner. Single cysteine variants K207C and M244C were generated as controls. The S235C/M244C variant performed as expected, decreasing the activity of the A170K variant to nearly background in the oxidized but not the reduced state (Supplemental Fig. 4). However, the M244C single mutant exhibited similar effects, suggesting that it forms disulfide bonds with cysteine(s) other than S235C. Indeed, the side chains of Cys200 and Cys234 are very close to that of M244C. The K207C/E251C mutant was similar to S235C/M244C under oxidized conditions, but ~15-fold more active (similar to WT DH/PH levels, see Fig. 3C) under reducing conditions. The K270C variant, on the other hand, exhibited higher activity than A170K on its own under oxidizing conditions, but similar activity to all the variants except K207C/E251C when reduced. These results suggest that K207C/E251C in a reduced state and K270C in an oxidized state favor a configuration where the DEP1 domain is less able to engage the DH domain and maintain the kinked state. The mechanism for this is not known. Regardless, these data show that perturbation of contacts between the kinked segments of the a6-aN helix can have profound consequences on the activity of the DH/PH-DEP1 core.”

    In the low-resolution cryo-EM study, it is mentioned that only a few classes exhibit the extra density that ultimately corresponds to autoinhibited P-Rex1. If so, is this also the case in the high-resolution study and how many of the most populated classes contribute to the autoinhibited structure? It would be informative for the reader to provide this information.

    Indeed, only a small subset of the particles are in the autoinhibited conformation in the Krios data set, similar to the Glacios. How many classes these particles partition to is dependent on how many classes are asked for during 2D classification and how many “garbage” particles are present at the different stages of particle stack cleaning during 2D classification. Also, because of the preferred orientation problem, many of the particles in this conformation segregate together during 2D classification. Therefore, in addition to the information show in Sup. Fig. 2, we think a more informative metric to answer the reviewer’s question is the number of particles at the start of data processing compared to at the end, which is shown in Table 1.

    Page 10, line 217: "The kink .... is important for autoinhibition". It seems unlikely that there is no kink in the activated state. Perhaps it should say something like "Mobility in the kink is important ..."

    Agreed. In fact, the SAXS data we reported on the DH/PH module in Ravala et al. (2020) is most consistent with a DH/PH that exhibits both extended and condensed conformations in solutions.

    Fig. 4A: It would help to label helices alpha6 and alphaN.

    These helices have now been labeled.

    Page 11, lines 223 and 228 are contradictory: In line 223 it is stated that K207C/E251C exhibit reduced GEF activity, while on line 228 it says this has little effect under non-reducing conditions.

    We thank the reviewer for this catch. We have modified the text to make it self-consistent.

    In Fig.5B, it would help if the authors mention in the legend that a trans-well migration assay was used, in order to know what the increase in stained cells signifies.

    The legend has been modified to include this information.

    The previous work by Chang et al., 2022 (PMID: 35864164) found that the final DH domain α6 formed the hinge helix (the kink in this manuscript), which undergoes a significant conformational change between closed and opened conformations of P-Rex1. Could the authors discuss the state of the kink in the presence of IP4 and in the P-Rex1 variants A170K and L177E?

    We have now included an alignment of our structure in the presence of IP4 with the Chang et al., 2022 structure (Supplemental Figure 5). There is very little difference in the kink region. Because the A170K variant exhibits reduced GEF activity and a smaller Dmax, it could be speculated that the kink might be further stabilized as compared to wild-type. The L177E variant exhibited activity similar to that of DH/PH alone, implying a relief of the kink. This interpretation is supported by our SAXS analysis of A170K and L177E in Fig. 3.

    I am a bit confused about the set of experiments with the intended DH-DEP1 interface disruptive mutation A170K, which later turned out to enhance P-Rex1 activity inhibition. The authors explained that the DH K170 salt bridges with DEP1 Glu411 stabilize the DH-DEP1 interaction. Next, the authors used P-Rex1 A170K mutant as the backbone for the introduction of disulfide bonds to block the closed configuration of the DH-PH hinge region by creating some mutants S235C/M244C and K207C/E251C. The first intended C235-C244 disulfide bond did not show any effect on the GEF activity because C235 is so close to the native C234 for a potential disulfide bond. I would recommend putting the data of S235C/M244C into a supplemental figure. Also, I am wondering if the GEF activity measurements in Fig 4B could be performed in the presence or absence of IP4 to see whether the IP4-induced autoinhibition form is distinct from the natural autoinhibitory once the kink was unblocked by reducing agent DTT.

    The confusion was warranted by our poor analysis of this data, rectified as discussed above.

    With regards to experiments plus/minus IP4, due to the absence of the IP4P domain, IP4 had no inhibitory effect on the activity of DH/PH or DH/PH-DEP1 (Supplemental Figure 1A and 1B) and as such this experiment would not likely be informative (or at best very hard to interpret).

    For the IP4 versus PIP3 activity assays, the authors indicated that P-Rex1 inhibition is dependent on the Inositol 3-phosphate. Have the authors tested and could they test with either Ins (1,3,4)P3 or Ins(1,3,5)P3?

    In these assays (Figure 1D), we show that inhibition does not occur with Ins(1,4,5)P3. Based on previous structures of IP4 bound to the PH domain and supporting biochemical assays (Cash et al., 2016, Structure), the 3- and 4-phosphates are the most highly coordinated and the next most thermostabilizing headgroup other than IP4 was Ins(1,3,4)P3. Therefore, we would anticipate that Ins(1,3,4)P3 might stabilize the autoinhibited state, perhaps at higher concentrations, but we have not directly tested this.

    The authors should provide the electron density maps of the P-REX1-IP4 complex in the supplemental figure and highlight the maps for two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle subdomain.

    The Coulomb potential map of this complex is shown in Figure 2A. Due to the moderate resolution of the reconstruction, side chain details cannot be unambiguously modeled at these interfaces, which is why we do not highlight any observed, specific interactions between sidechains.

    The manuscript was written very well and there is only one typing error in the legend of Supplemental Figure 1.

    Thank you for this catch.

    Details of EM density at significant domain interfaces and at the IP4 binding site should be provided as supplementary material.

    Beyond our comment about interfaces above, we have now provided the map representing the bound IP4 as Figure 4B.

    Line 123: It is difficult to discern in Figure 2A the "severe bend" in the helix that connects the DH and PH domains. It was not apparent (to me, at least) where this helix is located until eventually encountering Figure 4. It would be helpful to highlight or label (maybe with an asterisk) the bend site in Fig 2A.

    This has been labeled in Figure 2A.

    Line 125-126: likewise, It would be helpful to the reader to highlight the GTPase binding site in the DH domain.

    This has been labeled in Figure 2A.

    Line 159. Consider adding a supplementary figure showing a superposition of the two pREX-1 regulatory interfaces in the present structure and in 7SYF.

    A superposition of the two structures has now been added as Supplemental Figure 5. Because both structures are of moderate resolution, it is difficult to place side chains with a high degree of certainty. Thus, we did not think it wise to draw conclusions from comparisons between the details of these interfaces.

    Is the positioning of IP4 dictated by the EM density, prior knowledge from high-resolution structures, or both? A rendering of the EM density over the stick model as a supplementary figure would be helpful.

    This was modeled based on both. This image has now been added as Figure 4B.

    It should be emphasized that the jackknife model is similar to the hinge model proposed by Chang et al (2022).

    Mention of similarity between our model and the model proposed by Chang et al., 2022 occurs twice in the manuscript.

  3. eLife

    eLife assessment

    This important study contributes insights into the regulatory mechanisms of a protein governing cell migration at the membrane. The integration of approaches revealing protein structure and dynamics provides convincing data for a model of regulation and suggests a new allosteric role for a solubilized phospholipid headgroup. The work will be interesting to researchers focusing on signaling mechanisms, cell motility, and cancer metathesis.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation has not been explored.

    Strengths:

    The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound. The results are clear and support the conclusions.

    Weaknesses:

    (1) The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4 bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta-gamma is presented.

    (2) The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

    (3) Fig.1B-C: To give a standard deviation from 2 data points has no statistical significance. In this case it would be better to define as range/difference of the 2 data points.

  5. eLife

    Reviewer #2 (Public Review):

    Summary:

    In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibit form stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

    Strengths:

    The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain, and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gβγ subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

    Weakness:

    The set of experiments with the disulfide bond S235C/M244C caused a bit of confusion for interpretation, it should be moved into the supplement, and the text and Figure 4 were altered accordingly.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:

    In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access of the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

    Strengths:

    The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly-written discussion, the authors explain how PIP3, the cell membrane and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

    Manuscript revision:

    The reviewers noted a number of weaknesses, including error analysis of the HDX data, interpretation of the mutagenesis data, the small fraction of the total number of particles used to generate the EM reconstruction, the novelty of the findings in light of the previous report by Cheng et al, 2022, various details regarding presentation of structural results and questions regarding the interpretation of the inhibition data (Figure 1D). The authors have responded adequately to these critiques. It appears that pREX-1 is a highly dynamic molecule, and considerable heterogeneity among particles might be expected.

    While, indeed, the conformation of pREX presented in this report is not novel, the finding that this inactive conformational state is stabilized by IP4 is significant and important. The evidence for this is both structural and biochemical, as indicated by micromolar competition of IP4 with PI3-enriched vesicles resulting in the inhibition of pREX-1 GEF activity.

  7. Version published to 10.1101/2023.09.15.557836v3 on bioRxiv
  8. Version published to 10.1101/2023.09.15.557836v2 on bioRxiv
  9. Version published to 10.7554/elife.92822.1 on eLife
  10. Version published to 10.7554/elife.92822 on eLife
  11. eLife

    eLife assessment

    This important study contributes insights into the regulatory mechanisms of a protein governing cell migration at the membrane. The integration of approaches revealing protein structure and dynamics provides convincing data for a model of regulation and suggests a new allosteric role for a solubilized phospholipid headgroup. The work will be interesting to researchers focusing on signaling mechanisms, cell motility, and cancer metathesis.

  12. eLife

    Reviewer #1 (Public Review):

    Summary:
    The authors perform a multidisciplinary approach to describe the conformational plasticity of P-Rex1 in various states (autoinhibited, IP4 bound, and PIP3 bound). Hydrogen-deuterium exchange (HDX) is used to reveal how IP4 and PIP3 binding affect intramolecular interactions. While IP4 is found to stabilize autoinhibitory interactions, PIP3 does the opposite, leading to deprotection of autoinhibitory sites. Cryo-EM of IP4 bound P-Rex1 reveals a structure in the autoinhibited conformation, very similar to the unliganded structure reported previously (Chang et al. 2022). Mutations at observed autoinhibitory interfaces result in a more open structure (as shown by SAXS), reduced thermal stability, and increased GEF activity in biochemical and cellular assays. Together their work portrays a dynamic enzyme that undergoes long-range conformational changes upon activation on PIP3 membranes. The results are technically sound (apart from a few points mentioned below) and the conclusions are justified. The main drawback is the limited novelty due to the recently published structure of unliganded P-Rex1, which is virtually identical to the IP4 bound structure presented here. Novel aspects suggest a regulatory role for IP4, but the exact significance and mechanism of this regulation have not been explored.

    Strengths:
    The authors use a multitude of techniques to describe the dynamic nature and conformational changes of P-Rex1 upon binding to IP4 and PIP3 membranes. The different approaches together fit well with the overall conclusion that IP4 binding negatively regulates P-Rex1, while binding to PIP3 membranes leads to conformational opening and catalytic activation. The experiments are performed very thoroughly and are technically sound (apart from a few comments mentioned below). The results are clear and support the conclusions.

    Weaknesses:

    1. The novelty of the study is compromised due to the recently published structure of unliganded P-Rex1 (Chang et al. 2022). The unliganded and IP4-bound structure of P-Rex1 appear virtually identical, however, no clear comparison is presented in the manuscript. In the same paper, a very similar model of P-Rex1 activation upon binding to PIP3 membranes and Gbeta/gamma is presented.

    2. The authors demonstrate that IP4 binding to P-Rex1 results in catalytic inhibition and increased protection of autoinhibitory interfaces, as judged by HDX. The relevance of this in a cellular setting is not clear and is not experimentally demonstrated. Further, mechanistically, it is not clear whether the biochemical inhibition by IP4 of PIP3 activated P-Rex1 is due to competition of IP4 with activating PIP3 binding to the PH domain of P-Rex1, or due to stabilizing the autoinhibited conformation, or both.

    3. It is difficult to judge the error in the HDX experiments presented in Sup. data 1 and 2. In the method section, it is stated that the results represent the average from two samples. How is the SD error calculated in Fig.1B-C?

  13. eLife

    Reviewer #2 (Public Review):

    Summary:
    In this new paper, the authors used biochemical, structural, and biophysical methods to elucidate the mechanisms by which IP4, the PIP3 headgroup, can induce an autoinhibit form of P-Rex1 and propose a model of how PIP3 can trigger long-range conformational changes of P-Rex1 to relieve this autoinhibition. The main findings of this study are that a new P-Rex1 autoinhibition is driven by an IP4-induced binding of the PH domain to the DH domain active site and that this autoinhibited form is stabilized by two key interactions between DEP1 and DH and between PH and IP4P 4-helix bundle (4HB) subdomain. Moreover, they found that the binding of phospholipid PIP3 to the PH domain can disrupt these interactions to relieve P-Rex1 autoinhibition.

    Strengths:
    The study provides good evidence that binding of IP4 to the P-Rex1 PH domain can make the two long-range interactions between the catalytic DH domain and the first DEP domain and between the PH domain and the C-terminal IP4P 4HB subdomain that generate a novel P-Rex1 autoinhibition mechanism. This valuable finding adds an extra layer of P-Rex1 regulation (perhaps in the cytoplasm) to the synergistic activation by phospholipid PIP3 and the heterotrimeric Gbeta/gamma subunits at the plasma membrane. Overall, this manuscript's goal sounds interesting, the experimental data were carried out carefully and reliably.

  14. eLife

    Reviewer #3 (Public Review):

    Summary:
    In this report, Ravala et al demonstrate that IP4, the soluble head-group of phosphatiylinositol 3,4,5 - trisphosphate (PIP3), is an inhibitor of pREX-1, a guanine nucleotide exchange factor (GEF) for Rac1 and related small G proteins that regulate cell migration. This finding is perhaps unexpected since pREX-1 activity is PIP3-dependent. By way of Cryo-EM (revealing the structure of the p-REX-1/IP4 complex at 4.2Å resolution), hydrogen-deuterium mass spectrometry, and small angle X-ray scattering, they deduce a mechanism for IP4 activation, and conduct mutagenic and cell-based signaling assays that support it. The major finding is that IP4 stabilizes two interdomain interfaces that block access to the DH domain, which conveys GEF activity towards small G protein substrates. One of these is the interface between the PH domain that binds to IP4 and a 4-helix bundle extension of the IP4 Phosphatase domain and the DEP1 domain. The two interfaces are connected by a long helix that extends from PH to DEP1. Although the structure of fully activated pREX-1 has not been determined, the authors propose a "jackknife" mechanism, similar to that described earlier by Chang et al (2022) (referenced in the author's manuscript) in which binding of IP3 relieves a kink in a helix that links the PH/DH modules and allows the DH-PH-DEP triad to assume an extended conformation in which the DH domain is accessible. While the structure of the activated pREX-1 has not been determined, cysteine mutagenesis that enforces the proposed kink is consistent with this hypothesis. SAXS and HDX-MS experiments suggest that IP4 acts by stiffening the inhibitory interfaces, rather than by reorganizing them. Indeed, the cryo-EM structure of ligand-free pREX-1 shows that interdomain contacts are largely retained in the absence of IP4.

    Strengths:
    The manuscript thus describes a novel regulatory role for IP4 and is thus of considerable significance to our understanding of regulatory mechanisms that control cell migration, particularly in immune cell populations. Specifically, they show how the inositol polyphosphate IP4 controls the activity of pREX-1, a guanine nucleotide exchange factor that controls the activity of small G proteins Rac and CDC42 . In their clearly written discussion, the authors explain how PIP3, the cell membrane, and the Gbeta-gamma subunits of heterotrimeric membranes together localize pREX-1 at the membrane and induce activation. The quality of experimental data is high and both in vitro and cell-based assays of site-directed mutants designed to test the author's hypotheses are confirmatory. The results strongly support the conclusions. The combination of cryo-EM data, that describe the static (if heterogeneous) structures with experiments (small angle x-ray scattering and hydrogen-deuterium exchange-mass spectrometry) that report on dynamics are well employed by the authors

    Weaknesses:
    There are a few weaknesses. While the resolution of the cryo-EM structure is modest, it is sufficient to identify the domain-domain interactions that are mechanistically important, since higher-resolution structures of various pREX-1 modules are available.

  15. Version published to 10.1101/2023.09.15.557836v1 on bioRxiv