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Ahmadian, M. RHO GTPase-GDI Interactions. Encyclopedia. Available online: (accessed on 09 December 2023).
Ahmadian M. RHO GTPase-GDI Interactions. Encyclopedia. Available at: Accessed December 09, 2023.
Ahmadian, Mohammad. "RHO GTPase-GDI Interactions" Encyclopedia, (accessed December 09, 2023).
Ahmadian, M.(2021, December 01). RHO GTPase-GDI Interactions. In Encyclopedia.
Ahmadian, Mohammad. "RHO GTPase-GDI Interactions." Encyclopedia. Web. 01 December, 2021.
RHO GTPase-GDI Interactions

The RHO family GTPases, most prominently RAC1, CDC42, and RHOA, share two common functional characteristics, membrane anchorage and an on/off switch cycle.

CDC42 electrostatic steering G domain hypervariable region geranylgeranyl

1. Introduction

The RHO family GTPases, most prominently RAC1, CDC42, and RHOA, share two common functional characteristics, membrane anchorage and an on/off switch cycle [1]. They typically contain a conserved GDP/GTP binding domain, called G domain, and a C-terminal hypervariable region (HVR) ending with a consensus sequence known as CAAX (C is cysteine, A is any aliphatic amino acid, and X is any amino acid). Subcellular localization, which is critical for the biological activity of RHO GTPases, is achieved by a series of posttranslational modifications at the cysteine residue in the CAAX motif, including isoprenylation (geranylgeranyl or farnesyl), endoproteolysis and carboxyl methylation [2]. Membrane-associated RHO GTPases act then, with some exceptions [3], as molecular switches by cycling between an inactive GDP-bound state and an active GTP-bound state. This cycle underlies two critical intrinsic functions, GDP-GTP exchange and GTP hydrolysis, which induce structural rearrangements of two regions of the protein, called switch I and switch II [3] (encompassing amino acids 29–42 and 62–68, respectively) and is controlled by two classes of regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) [4]. RHO GTPases act as dynamic switches in many developmental and cellular contexts [5] by selectively binding to and activating structurally and functionally diverse effectors. This class of proteins activates a wide variety of downstream signaling cascades [6][7][8][9], thereby regulating many important physiological and pathophysiological processes in eukaryotic cells [10][11][12].
The spatial and temporal activation of RHO GTPases inside a cell is fundamental, for example, to the regulation of local movements and cell-cell contacts that are required for morphogenesis [12]. They are commonly found to cycle between two pools, a membrane-associated and a cytosolic pool. Given the fact that membrane attachment is a prerequisite for the signaling roles of this protein family, it is clear that reversible membrane translocation offers cells a means to regulate the location of the activation event. However, there is a serious handicap to such physical cycling for RHO GTPases. The highly hydrophobic geranylgeranyl (GG) moiety of RHO proteins renders them energetically unfavorable to partition into the cytosol as individual monomers. Post-translationally modified RHO proteins can only detach from membranes if they are assisted by RHO-specific guanine nucleotide dissociation inhibitors (GDIs), that shield the bulky lipid moieties from the aqueous environment of the cytosol [4][13].
In contrast to the tremendous number of the other regulatory proteins of the RHO family (74 GEFs and 66 GAPs) [14][15], only three GDIs exist in the human genome [16]. The GDI family includes the ubiquitously expressed GDI1 (or GDIα) [17], GDI2 (GDIβ, LY-GDI or D4-GDI), which is mainly found in hematopoietic tissue, particularly in B- and T-lymphocytes [18], and GDI3 (or GDIγ) that is preferentially expressed in brain, pancreas, lung, kidney, and testis [19]. Unlike the other two GDIs, GDI3 contains an N-terminal extension that confers anchorage into the membranes of Golgi vesicles [20]. As GDI3 is very similar to GDI1, it can form a complex with all the GDI1 targets [21]. GDI1 and GDI2 contain at their very N-terminus a large number of acidic residues which have been proposed to be essential for their function in the cell [22]. In addition to their physiological expression, GDIs are expressed in several human cancers, including breast, liver, ovarian, pancreatic and myeloid leukemia [13][18][23][24]. Changes in GDI expression levels have shown pro-tumorigenic or anti-tumorigenic effects, that are cell type- and tissue-dependent. One reason for these opposite effects is most probably due to our lack of understanding of the basic mechanism of GDI function and their binding specificities to the different RHO proteins.
Understanding the mechanisms by which signaling events are localized and the physiological consequences of spatial restriction are exerted, is one of the major challenges in cell biology. Comprehensive studies in the last three decades have provided insight into the structure and function of these regulators acting as a shuttle for the RHO GTPases [13][25][26][27]. The shuttling process, which considerably differs from the KRAS4BFar-PDEδ [28][29][30][31], involves the extraction of RHO GTPases from donor membranes, formation of cytosolic GDI-RHO GTPase complexes and delivery of RHO GTPases to the target membranes [13][27]. Accordingly, it has been proposed that GDI regulates the isoprenylation process in the cell [32]. GDI is known to extract RHO GTPases from the membrane, maintain them in an inactivated state, and protect them from both degradation and unspecific activation by RHO-specific GEFs [13][33][34]. Structural studies by different groups have revealed two sites of interaction between GDI and RHO GTPases [35][36][37][38][39].

2. Geranylgeranyl Moiety Is Dispensable for RAC1-GDI1 Interaction

To understand the impact of the isoprenyl moiety of RAC1 on GDI1 binding, we compared the biochemical properties of geranylgeranylated RAC1 (RAC1GG) from insect cells and non-isoprenylated RAC1 full-length (RAC1FL) from Escherichia coli. Previous mass spectrometric analysis and liposome sedimentation of intact RAC1GG, compared to RAC1FL, have revealed a fully modified RAC1GG by geranylgeranylation [34]. Unless otherwise stated, the unmodified RAC1 purified from E. coli is designated as RAC1FL, the modified RAC1 purified from insect cells as RAC1GG, and the unmutated (wild-type) RAC1 as RAC1WT in cell-based experiments.
We determined the GDI association rates with both RAC1GG and RAC1FL using a stopped-flow fluorometric assay. Figure 1A shows a rapid decrease in fluorescence after mixing GDI1 with the RAC1 proteins, which is directly related to the association reaction between the RAC1-GDI1 pairs. Observed rate constants (kobs) obtained by a single exponential fitting increased linearly as a function of the GDI1 concentrations (Figure 1B), and yielded similar association rate constants (kon) for both RAC1GG and RAC1FL. The dissociation of the GDI1 from mdGDP-bound RAC1 proteins was measured in a displacement experiment. Observed single exponential fluorescence increase yielded respective dissociation rate constants (koff), which differ only 3-fold (Figure 1C). Notably, the RAC1GG-GDI1 interaction showed a biphasic behavior (double exponential kinetics); particularly for the off-rate, an initial rapid increase in fluorescence was followed by a slow plateau phase, which can be attributed to the GG moiety of RAC1GG. Calculated dissociation constants (Kd) from the ratio of the koff and kon values (Figure 1D) unexpectedly revealed only a 7-fold higher affinity for RAC1GG vs. RAC1FL.
Figure 1. RAC1GG and RAC1FL bind with a similar affinity to GDI1. (A) Association of GDI1 (4 µM) with RAC1GG and non-isoprenylated RAC1FL (0.2 µM, respectively). (BD) Quantitative measurements of GDI1 interaction with RAC1GG and RAC1FL led to the calculation of the individual binding constants, association rate constant or kon (B), dissociation rate constant or koff (C), and dissociation constant or Kd directly from the koff/kon ratio (D). (E) Titration of increasing GDI1 concentrations to RAC1GG and non-isoprenylated RAC1FL (0.2 µM, respectively), using fluorescence polarization, resulted in the determination of the equilibrium Kd values.
Instead of the simple exponential decrease seen with GAP, there is an initial rapid increase in fluorescence followed by a decrease at a rate similar to that seen at high GAP concentrations. The first phase of the reaction is concentration-dependent, whereas the second is less obviously so. This suggests that an initial association between the proteins leads to an increase in fluorescence, which then decreases on mant-GTP hydrolysis (and consequent dissociation of the proteins) to a level below that of the starting level, in agreement with the observation that the fluorescence of Ras‚mant-GDP is lower than that of Ras‚mant-GTP. This interpretation means that the mechanism cannot be treated as a very rapid initial equilibration followed by a relatively slow cleavage step, which is the underlying assumption in the analysis of the data obtained with GAP.
This result clearly contradicts existing models which have suggested that the isoprenyl moiety of the RHO GTPases contributes to orders of magnitude higher binding affinity for GDI as compared to unmodified RHO GTPases [40][41]. The studies have determined Kd values of 0.4 nM and 5 pM for the interaction of GDI1 with prenylated RAC1 and RHOA, respectively, as compared to the 180 nanomolar Kd value determined in our study. The reason for the extraordinarily large differences between the binding affinities obviously lies in the proteins and the type of fluorescence reporter groups used in each case. On the one hand, we and Tnimov et al. applied native GDI1 while Newcombe et al. [40] used a coumarin-labeled GDI1 at position cysteine 79, which is actually buried and located in the back side of the C-terminal geranylgeranyl binding domain (GGBD); this modification at this position can drastically alter the confirmation and consequently its biochemical properties of GDI1. On the other hand, native RAC1GG purified from insect cells was used by Newcombe et al. as well as in this study, Tnimov et al. [41] used a cell-free modification approach of RHOAFL and purified geranylgeranyl transferase I and nitrobenzoxadiazole (NBD)-labeled geranyl-pyrophosphate [1]. In this way, the nature of the isoprenyl moiety and the absence of further posttranslational modifications by specific proteolytic removal of the terminal three residues, and carboxylmethylation of prenylted cysteine residue, are largely different from native RHOAGG.

3. Conserved G Domain Is Not Rate-Limiting for the GDI1 Binding

The above findings prompted us to unambiguously challenge the paradigm of RAC1 regulation by GDI1. To this end, we investigated the interactions of the three GDI paralogs, GDI1, GDI2 and GDI3, with various non-isoprenylated members of the RHO GTPase family. GDI3 was purified as an N-terminal truncated variant lacking the amphipathic helix (amino acids or aa 1-20). Kinetics of association of the GDIs (2 µM) with 0.2 µM mdGDP-bound RHO GTPase was monitored under the same conditions as described above for RAC1. Calculated kobs values for each measurement were plotted as bar charts in Figure 2A, which clearly show that all three GDIs associated with RAC1, RAC3, RHOG and RHOA under the experimental conditions but apparently not with RAC2, CDC42, TC10, TCL, RHOB, RHOC, RHOD and RIF. GDI2 and GDI3 showed significantly faster association with RAC3 as compared to GDI1. Most remarkably, unlike GDI1 and GDI3, GDI2 was able to bind RAC2. These results clearly indicate for the very first time that GDIs can discriminate between the RHO GTPases by interacting with some but not all the RHO proteins.
Figure 2. Biochemical and structural view into the RHO GTPase-GDI interactions. (A) Kinetics of association of 2 µM GDI proteins (GDI1, GDI2 and GDI3ΔN20) with 0.2 µM mdGDP-bound RHO GTPases (twelve different proteins) was only monitored for RAC1, RAC3, RHOG and RHOA using stopped-flow fluorimetry. GDI2 but not GDI1 and GDI3 associated with RAC2. No binding was observed (n. b. o.) for the other GTPases. Obtained kobs values are the average of four to six independent fluorescence measurements, consisting of 1000 data points each (mean ± S.D.). (B) Individual rate constants were determined, under the same conditions as shown in Figure 1A–C, for the interaction of GDI1 with RAC1, RAC3, RHOG and RHOA, and GDI2 with RAC1, RAC2 and RAC3, respectively. Kinetic data were derived from the average of four to six independent measurements (mean ±S.D.). (C) An interaction matrix of the GDI proteins with twelve RHO family GTPases is generated to determine the frequency of contacts in respective structures. It comprises the amino acid sequence alignments of the RHO proteins (lower left panel) and the GDIs (upper right panel), respectively. Each element corresponds to a possible interaction of RHO residues (row; RAC numbering) and GDI residues (column; GDI1 numbering). The number of actual contact sites between RHO and GDI proteins (with distances of 4 Å or less) were calculated and are indicated with numbers for matrix elements between 1 and 9. (D) A detailed view into the structure (PDB code: 1HH4) of GDP-bound RAC1GG (grey ribbon) in complex with GDI1 (surface representation) revealed that the basic HVR (blue) is sandwiched between a series of acidic residues of GDI1 supplied by NTA (purple) and GGBD (orange). (E) Schematic diagrams of the domain organizations of GDI1 and RAC1GG illustrate their detailed boundaries. Amino acid sequence alignments of the N-terminal arm (NTA; 25 amino acids) and the C-terminal six residues of the GDI proteins (boxed) highlight negatively and positively charged residues (red and blue). Colors are the same in (D).
To examine binding properties, the respective association rate constants (kon) and the dissociation rate constants (koff) were determined for the interaction of GDI1 with RAC1, RAC3, RHOG and RHOA, and GDI2 with RAC2 under conditions described above (Figure 1A–D). All kinetic parameters along with calculated dissociation constants (Kd) are summarized in Figure 2B. The data are very similar for the GDI1 interaction with RAC1, RAC3, RHOG and RHOA with Kd values between 0.9 and 3.2 µM. However, GDI2 interactions exhibited similar rate constants for RAC1 and RAC3, which were significantly different from that of GDI1 (Figure 2B). GDI2 exhibited more than 10-fold faster kon values and up to 4-fold slower koff values. GDI2-RAC2 interaction was characterized by a much slower rate of association as compared to RAC1 and RAC3, resulting in a binding affinity of 18.8 µM under the given experimental conditions (Figure 2B).
The biochemical characterization together with structural studies has shown that the RAC paralogs exhibit different properties concerning ligand– and protein–protein interactions [42]. Whereas RAC1 and RAC3 behave almost identically, RAC2 revealed a 25-fold lower nucleotide affinity because of a decreased nucleotide association rate, a slightly higher PAK1 (p21 activated kinase-1, a downstream effector for RAC1 and CDC42) binding affinity, and a significant increase in GEF-catalyzed nucleotide dissociation. These aberrant properties most likely are the consequence of different conformational flexibilities in the switch I region [43].
A comparison of available structures of the RAC1-GDI1 and RAC2-GDI2 complexes revealed a rather high sequence similarity [35][36]. An inspection of full interaction matrix revealed very few amino acid deviations within the RAC G domains (Y/F89) and GDI paralogs (A/P/G31, E/K/R53, A/T/V54). However, Y98 in RAC1 and RAC3 undergoes contacts with H23 and V25 of GDI1 which may not be achieved by F89 in RAC2, that may only contact V25 but not S23. Among the deviations in GDIs, the loop containing A/G31 in GDI1 and GDI3 is in close vicinity of D65/R66 of RAC paralogs, which may, in the case of P31 in RAC2, adopt a different orientation and thereby influence RAC2-GDI2 binding. Residues in SWBD, such as E53 and A54 (GDI1 numbering), appear to be critical for RHO GTPase-GDI interaction. The double mutation of L55/L56 to serines in GDI1 has been shown to drastically decrease its affinity for RAC1 [44].

4. RAC1 Polybasic Motif Dictates GDI1 Binding

A sequence analysis of HVRs of GDI1 associating RHO GTPases (‘binders’) versus those with no observed GDI1 association (‘non-binders’) showed clear differences in both numbers and relative positions of positively charged residues (Figure 3A). To examine the impact of HVR on the RHO GTPase-GDI1 interaction, we measured the kinetics of GDI1 association with different HVR variants of RAC1 and RAC2. Remarkably, a loss of RAC1 association was observed with a C-terminal truncated variant lacking HVR-CAAX (RAC1ΔC10) as well as KRKRK-to-EEEEE (RAC15xE; charge-reversal variant) and KRKRK-to-QQKRA (RAC1-to-RAC2 or RAC1RAC2 variant). In contrast, a gain of GDI association with RAC2 was observed with QQKRA-to-KRKRK (RAC2-to-RAC1 or RAC2RAC1 variant; Figure 3B). These findings were verified by fluorescence polarization experiments and obtained data summarized in Figure 3C revealed that (1) RAC1ΔC10 yet bound GDI1 with a 26-fold lower affinity as compared to RAC1, (2) RAC15xE, binding to GDI1 was yet observed with a very low affinity while this was not possible for RAC1RAC2, and (3) RAC2RAC1 did, in contrast to RAC2, bind GDI1 with an almost similar affinity as determined for RAC1. Taken together aided HVR alteration can completely abolish GDI1 association with RAC1 and revert GDI1 association with RAC2.
Figure 3. RAC1 HVR generates a selective and high affinity interaction toward GDI1. (A) A sequence alignment of hypervariable region (HVR) of RHO GTPases shows significant differences in the frequency of the basic residues (blue). GDI-binding proteins are shown in bold. Critical amino acid deviations in RAC2 are shown in orange. The isoprenylation site (cysteine 189 in RAC1) is highlighted in bold. (B) Kinetics of GDI1 association were measured by mixing RAC1 and RAC2 variants (0.2 µM, respectively) with 2 µM GDI1. (C) Kd values for the RHO GTPase-GDI1 interaction were determined by titrating RAC1 and RAC2 variants (0.2 µM, respectively) with increasing concentrations of GDI1 using fluorescence polarization.
The results clearly demonstrate the critical role of the polybasic motif of RAC1 in determining GDI1 binding. It seems that both an increase of overall positive charge in HVR of RAC2 and the distance of the basic residues from the geranylgeranyl site strongly reinforce GDI1 binding affinity. We hypothesize that the GDI1 selectively binds RAC1 polybasic motif to pull the GG moiety from the plasma membrane and direct it into the hydrophobic cavity of its GGBD.
The relative position and the order of the basic residues in HVR seem to contribute to the formation of an electrostatic network (Figure 2E) that may significantly stabilize GDI1 interaction with, for example, RAC1 and RAC3 but not RAC2. Synthetic peptides containing the polybasic motifs of RAC1 (aa 178–188), but not RAC2 (178–188), have been shown to inhibit NADPH oxidase activity in a RAC1-dependent system, and interfere with the translocation of RAC1 proteins to the plasma membrane [45]. While the geranylgeranyl moiety mediates membrane anchorage, the polybasic motif of RAC1 interacts with plasma membrane phosphoinositides and stabilizes its proper orientation [46].
Considering the amino acid sequence identity of the G domain on the one hand (Figure 2C), and the sequence similarities among the hypervariable regions on the other (Figure 3A), it is striking that seven out twelve RHO family GTPases do not interact with GDI1 (Figure 2A). For example, the HVRs of RHOA versus RHOC and RAC3 versus CDC42 look very similar, and yet GDI1 binds one but not the other, under the same experimental condition in this study. An in-depth analysis of the RAC1 and RAC2 variants revealed that the HVR polybasic motif dictates GDI1 binding (Figure 3B,C). The number of positively charged residues appears not to be a binding determining factor since the HVR polybasic motif of the nonbinder RHOC has a higher positive net charge as compared to the binder RHOA (Figure 3A). This is also true if comparing the binder RAC3 with the nonbinder CDC42, which exhibits a much larger amino acid variability with their HVRs. Thus, we assume at this stage that, not the number of positive charges but rather the position of the basic residues relative to the C-terminal cysteine determines the bilateral binding selectivity of the HVR polybasic motif by the negatively charged residues of both GGBD and NTA (Figure 2D,E). This may be the reason for TC10 and RHOD not to interact with GDI1 related to the distance of the polybasic motif to the C-terminal Cysteine (Figure 3A). Notably, Gosser et al. has reported a binding affinity of 1.6 nM between unmodified CDC42 and GDI1, which significantly impaired upon N-terminal deletions of GDI [47]. This value is three orders of magnitude lower than the Kd values we have obtained from kinetic and equilibrium measurements (Figure 1). Gosser et al. have used in addition to mGDP-bound CDC42 also a fluorescein-conjugated GDI at position cysteine 79, which is actually buried and located in the back side of the GGBD; this modification at this position can drastically alter the confirmation and consequently int biochemical properties of GDI1. Moreover, residues next to the positively charged residues within the HVR seem to play a role, too. Glutamines in RAC2 and RIF seem to be deleterious for the interaction with GDI1. Glutamate 181 in CDC42 may exert electrostatic repulsive effects on the GDI binding (Figure 3A). Serine 185 is a phosphorylation site on CDC42, regulating its translocation to the cytosol by favoring its interaction with GDI1 [48]. Future studies will shed light on these issues.

5. Electrostatic Pincer Residues of GDI1 Grasp RAC1 HVR

GDI1 function appears to be driven and controlled by electrostatic forces, that attract the polybasic motif of RAC1. To examine this mechanism, we generated different deletion and charge reversal variants of GDI1 and measured both their binding capabilities to RAC1GG and RAC1FL, as well as their functional properties to displace RAC1GG from PIP-enriched synthetic liposomes. GDI1E121K, which apparently does not contact RAC1 HVR but the switch 2 region (Figure 2E), was used as a control. Kinetic analysis showed that most GDI variants are disabled in associating with RAC1 (Figure 4A). Substitutions of D140, E163 and E164 for lysines or deletion of the N-terminal and very C-terminal amino acids significantly impaired GDI1 binding to isoprenylated and non-isoprenylated RAC1, as compared to GDI1WT. The most drastic effects were observed with 25 and 58 amino acids deleted at the N-terminus (∆N25 and ∆N58) on the one hand, and double (E163K and E164K or 2E > 2K) and triple (E140K, E163K and E164K or 3E > 3K) mutations, on the other, which did not bind to the RAC proteins under the experimental conditions. Fluorescence polarization measurements verified that most GDI1 variants were yet able to bind RAC1FL, but with up to 145-fold lower binding affinities compared to GDI1WT (Figure 4B). RAC1 binding was completely abolished in the case of ∆N58 and 3E > 3K variants. GDI1ΔN58 not only lacks the very N-terminal acidic resides, that are integral elements of the electrostatic pincer function, but also the switch binding domain (SWBD), which forms multiple contacts with the RAC1 switch regions (Figure 2C,D). GDI13E>3K most likely creates intermolecular charge repulsion towards positively changed HVR.
Figure 4. RHOGDI1 grasps basic HVR of RAC1 with multiple negatively charged residues. (A) Kinetics of association of 2 µM GDI1 variants with 0.2 µM mdGDP-bound RAC1 was monitored using stopped-flow fluorimetry. The obtained data are the average of four to six independent measurements (mean ± S.D.). (B) Kd values for the interaction between the GDI1 variants and mdGDP-bound RAC1 were determined by fluorescence polarization. (C) Individual rate constants were determined, under the same conditions as shown in Figure 1A–C, for the interaction of RAC1 with GDI1 WT, ΔN15 and ΔC6, respectively. Kinetic data were derived from the average of four to six independent measurements (mean ± S.D.). (D) GDI1 variants are, in contrast to GDI1WT, impaired in extracting RAC1GG from PIP-enriched synthetic liposomes. The graphs represent densitometric analysis of three independent liposome sedimentation experiments. Data are expressed as the mean of triplicate experiments ± standard deviation (unpaired t-test, * p < 0.05, ** p < 0.01, and *** p < 0.001). (E) FLAG-GDI1 variants were not able to extract YFP-RAC1 from the plasma membrane of HUVECs. Scale bar represents 50 µm. Arrow point to colocalization of RAC1 and GDI1 at the membrane ruffles.
To analyze the function of the GDI variants in extracting RAC1GG from the liposomes, we performed a liposome sedimentation assay established previously [34]. Therefore, we mixed PIP-enriched liposomes (200 µm in diameter) with GDP-bound RAC1GG and isolated liposome-bound RAC1GG from the pellet fraction (Figure 4D, first lane) after sedimentation. Next, 1 µM of RAC1GG•GDP bound to liposomes was mixed with 2 µM of GDI1 WT and its variants (2 µM, respectively) to measure their ability to displace RAC1GG from the liposomes. Figure 4D shows that GDI1WT quantitatively displaced RAC1GG from the liposomes. In contrast, the majority of the GDI1 variants revealed a significant reduction in their activities, consistently with the kinetic and equilibrium measurements (Figure 4A–C). Particularly, GDI1ΔN58 and GDI13E>3K were completely disabled in binding and extracting RAC1GG from the liposomes, strongly supporting the notion that GDI1 supplies an electrostatic pincer to grasp RAC1 and pull it out from the plasma membrane. Moreover, GDI1E121K and GDI12E>2K remained partially associated with liposomes and were sedimented in the pellet fraction (Figure 4D). The fact that these GDI1 variants were able to bind to RAC1GG on the liposomes but could not extract it from the liposomes strongly suggests that an electrostatics-guided binding and extraction mechanism is impaired unilaterally. We think that binding of the GDI1 GGBD with the RAC1 HVR takes it away from its membrane association and release additional basic residues on HVR for the interaction with the negatively charged residues of the GDI1 NTA (the so-called electrostatic pincer; Figure 5). Loss of the GGBD-HVR interaction at this step obviously disabled the GDI1 variants (GDI1E121K and GDI12E>2K), that is associated with RAC1GG on the liposomes, to extract RAC1GG from the membrane.
Figure 5. Schematic models of GDI1-regulated RAC1 extraction from the cell membrane. (A) GDI1 SWBD (green) recognizes and binds the switch regions of RAC1 to initiate its extraction from the membrane. (B) This step is followed by the electrostatic attraction of RAC1 polybasic region through the negatively charged GGBD (orange) and probably also NTA (purple). (C) NTA and GGBD are integral elements of the electrostatic pincer function, creating intermolecular charge attraction forces towards positively changed HVR. (D) This electrostatic steering mechanism rounds off the GDI1-mediated RAC1 extraction from the membrane by locking the geranylgeranylated C-terminus of RAC1 through the terminal charged regions of GDI1, and safeguarding RAC1GG -bound state of the GDI. Coloring is the same as the coloring of GDI1 and RAC1 structures shown in Figure 2D,E. Positive charges are schematically shown as + and negative electrostatics as −. For more details, see the discussion and conclusions.


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