## 2 Results and Discussion
### Q61L Variant Is Not a Wildtype Equivalent for CDC42-IQGAP Interactions  
An issue that still needs to be addressed is why are there several discrepancies between the studies regarding the GRD binding property for CDC42? A possible answer to this question is the use of different CDC42 mutants in these studies that are alike, but not equivalent, especially regarding this interaction. Figure 3: CDC42 IH mutations decrease binding affinity. (**A**) Amino acid alignment of the insert helix of selected members of the RHO GTPase family. CDC42 mutations (red) to RAC1 were introduced outside of the conserved regions (grey). (**B**) Fluorescence polarization data for the interaction of GRD1 and C794 with the CDC42 IH variants. (**C**) Pull-down of endogenous IQGAP1 FL from HEK293 lysates with GST-CDC42 IH variants in both GppNHp-bound and GDP-bound forms. Cell lysate was used as an input control. The pull-down data for GST-CDC42 wt is shown in Figure 2F as all pull-down experiments were conducted under the same conditions. Densiometry evaluation of relative IQGAP1 binding to GST-CDC42 proteins (a. u., arbitrary unit) from a triplicate experiment is shown as bar charts. In the GTP-bound CDC42, Q61 acts as a 'catalytic residue' that is involved in hydrogen bonding with a catalytic water molecule, an arginine finger of GAP and the \(\gamma\)-phosphate of GTP, initiating a nucleophilic attack that hydrolyzes GTP (Figure 4A) [18, 65]. L61 does not, however, undergo these functionally critical hydrogen bonds but rather points towards protein surface without causing significant structural changes (Figure 4A). As a result, the substitution of Q61 by leucine drastically increases the binding affinity of IQGAPs for CDC42\({}^{\text{Q61L}}\) GTP by up to 15-fold as was clearly demonstrated previously [5, 26, 51]. Despite this fact, many groups use this CDC42 variant for the interaction analysis of effectors, such as IQGAPs [12, 54, 56, 57]. Thus, we revisited this issue and have comparatively analyzed the interaction of IQGAP1 GRD with CDC42\({}^{\text{Q61L}}\) and CDC42\({}^{\text{wt}}\) using fluorescence polarization and size exclusion chromatography (SEC). Equilibrium measurements shown in Figure 4B clearly revealed that the Q61L mutation results in a strong enhancement of GRD1 and GRD2 binding with the mGppNHp-bound CDC42, but not with mGDP-bound CDC42. The binding affinity of mGppNHp-bound CDC42\({}^{\text{Q61L}}\) rises from a low affinity 186 uM/69 uM binding to a high 2.7 uM/2.5 uM binding for GRD1/GRD2, respectively (Figures 4B and S7). This is a change of 30-50-fold and might explain the huge differences of CDC42 interactions with GRD. Moreover, SEC analysis showed that GRD1 forms a 2:2 stoichiometry with CDC42\({}^{\text{wt}}\) GppNHp but 2:1 stoichiometry with CDC42\({}^{\text{Q61L}}\) GppNHp (Figure 4C-F).The latter is remarkably consistent with the previous reports on a high-affinity binding of IQGAP2 GRD2 with CDC42\({}^{\text{Q61L}}\) GTP and 4:2 and 2:1 stoichiometry, respectively [12, 54]. These findings verified the clear difference between CDC42\({}^{\text{wt}}\) and CDC42\({}^{\text{Q61L}}\) and how replacement of Q61 by L changes the binding properties (affinity and stoichiometry) of CDC42 interaction with IQGAP GRDs. [PERSON] et al. have reported that the Q61L mutation strengthen hydrogen bond interactions between CDC42 and the \(\gamma\)-phosphate of GTP [66]. Analyzing the Cdc42\({}^{\text{Q61L}}\) GTP GRD2 structure, [PERSON] et al. proposed that CDC42 IH binding to the GAPex-domain induces allosteric changes in the RASGAP site, which in turn facilitate GRD dimerization, and enable the second CDC42\({}^{\text{Q61L}}\) to bind to this site (yielding a 2:1 stoichiometry) [54]. Collectively, we recapitulate that CDC42\({}^{\text{Q61L}}\) is not an ideal analog of CDC42\({}^{\text{wt}}\) especially in studying the interaction of the downstream effectors. G12V and Q61L mutations of CDC42 cause GAP insensitivity leading to sustained hyperactivation of CDC42 [16, 18, 55, 65, 66].