## 2 Results and Discussion
### GRD Lacks the Structural Fingerprints to Induce the GAP Activity  
The structure of the RAS-RASGAP complex shows GAP-334 interacting predominantly with the switch regions of RAS [55]. Three regions (finger loop, FLR motif and helix \(\alpha\)/variable loop) constitute structural fingerprints of the RASGAP p120 and neurofibromin that form critical RAS binding sites in order to apply an arginine finger into the active center of RAS [67, 68]. Amino acid sequence analysis of these RASGAPs with the three IQGAP paralogs showed that major parts of these fingerprints are different in IQGAPs (Figure 5A). Moreover, the catalytic arginine is missing and there is instead a threonine (T1045 in IQGAP1; Figure 5A). Thus, it is quite understandable why IQGAP1 did not display RASGAP activities towards HRAS [60]. It is, however, known that GAPs specific for other members of the RAS superfamily use other catalytic residues than an arginine (reviewed in [69, 70]). We set out to examine a possible GAP activity of IQGAP1 GRD towards different RAS family GTPases. Figure 5B shows that IQGAP1 GRD is a pseudo-RASGAP domain with no obvious catalytic ability (orange bars). Earlier studies have shown that the substitution of the arginine finger of the RASGAPs to other amino acids completely abolishes their GAP activity [67, 68]. Therefore, threonine 1046 of IQGAP1 GRD was replaced by an arginine and the impact of T1046R on the GTP hydrolysis of the eight RAS proteins was measured. Data shown in Figure 5B revealed no apparent GAP activities of IQGAP1 GRD\({}^{\text{T1046R}}\) (green bars)as expected for a RASGAP. These data suggest that IQGAPs, besides lacking an arginine finger, do not contain critical RAS-binding residues of the a7/variable loop (Figure 5A). Figure 4: Comparative analysis of IQGAP1 GRD1 interaction with CDC42\({}^{\text{Q61L}}\) and CDC42\({}^{\text{wt}}\). (**A**) Structural overlay of CDC42\({}^{\text{wt}}\) GDP AIF\({}_{3}\) p50 GAP (green; PDB: 1 GRN) on CDC42\({}^{\text{Q61L}}\) GTP IQGAP2 (blue; PDB: 5 CJP) with the focus on Q61 hydrogen bonds (red dashed lines). GDP AIF\({}_{3}\) mimics the transition state of the GTP hydrolysis reaction and is coordinated with the magnesium ion (Mg\({}^{2+}\)) and the nucleophilic water molecule (w) and the arginine finger (R282) of p50 GAP. Aluminum trifluoride (AIF\({}_{3}\)) mimics the \(\gamma\)-phosphate of GTP in the transition state. In contrast to L61, Q61 is critical for the catalysis of the GTP hydrolysis reaction through three hydrogen bonds (see text). (**B**) Fluorescence polarization data of IQGAP GRD1 with CDC42 mGppNHp and CDC42 mGDP. (**C**–**F**) IQGAP GRD differently forms complexes with CDC42\({}^{\text{WT}}\) and CDC42\({}^{\text{Q61L}}\), respectively, when applied on an analytical SEC. For this purpose, CDC42\({}^{\text{WT}}\) GppNHp (**C**) or CDC42\({}^{\text{Q61L}}\) GppNHp (**D**) were mixed with IQGAP1 GRD1 and SEC was performed on a Superdex 200 10/300 column using an AKTA purifier (flow rate of 0.5 mL/min, fraction volume of 0.5 mL) and a buffer, containing 30 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 5 mM MgCl\({}_{2}\). The elution profiles represented one peak for the respective CDC42 proteins (#1), two peaks for the respective mixtures of respective CDC42 proteins with GRD (#2 and #3) and one peak for the GRD1 (#4). (**E**) Coomassieie brilliant blue staining of the corresponding elution volumes indicated that only peaks #2 contain GRD1 complexes with CDC42\({}^{\text{WT}}\) or CDC42\({}^{\text{Q61L}}\), respectively. Peaks #3 only contain the CDC42 proteins as compared to the peaks #1 and #4. (**F**) The SEC profiles of CDC42\({}^{\text{WT}}\) and CDC42\({}^{\text{Q61L}}\) are summarized for each peak regarding the elution volume, the molecular weight (MW) and the stoichiometry. M stands for monomeric and D for dimeric. The theoretical MWs of CDC42 (21.2 kDa) and GRD (43 kDa) were calculated using the Expasy Protparam tool.