Selectivity Determinants of RHO GTPase Binding to IQGAPs

Abstract

IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.

Keywords: CDC42; IQGAP; RAC1; RHO GTPases; scaffold; selective bindings.

Figure 1

IQGAP1 and IQGAP2 selectively associate with CDC42 and RAC1-like proteins. (A) Domain organization of the IQGAP paralogs and their C-terminal fragments assessed in this study (see text for more details). (B) The association of IQGAP1^C794 and IQGAP2^C795 (2 µM) with various mGppNHp-bound RHO GTPases (0.2 µM) was investigated (Figure S1). The k_obs values for the interaction of IQGAP1 and IQGAP2 with several RHO GTPases, shown as bars, illustrate that both IQGAPs associate with CDC42 and RAC1-like proteins. The RHO-like proteins RND1, RND2, RND3, TC10, RIF, and RHOD did not associate with these IQGAPs under these conditions. (C) The association rates (k_on) were measured using 0.2 µM mGppNHp-bound RHO GTPases with increasing concentrations (2–8 µM) of IQGAP1^C794. Dissociation rates (k_off) were measured by mixing 2 µM IQGAP1^C794 complexed with mGppNHp-bound RHO GTPases (0.2 µM) and unlabeled RAC1-GppNHp (10 µM). The individual rate constants were calculated for the interaction of IQGAP1^C794 with RAC- and CDC42-like proteins, and the results are plotted in bar charts. Association rates (k_on), dissociation rates (k_off), and dissociation constants (K_d) for IQGAP1^C794-RHO protein binding are shown. RAC2 showed the highest binding affinity for IQGAP1^C794, followed by CDC42, RAC3, RHOG, and RAC1. The data are expressed as the means ± S.D. All measurements were obtained in duplicate. n. s. o. = no signal observed. Kinetic data, which are summarized in Table S1 and shown in Figure S2, were obtained in triplicate. The data are expressed as the means ± S.D. (D) Binding of endogenous IQGAP1 to GppNHp-bound RHOG and CDC42 (left panel) was analyzed in a GST pull-down assay (n = 3) using total cell lysate (TCL) of HEK-293 cell (i, input; o, output). GST-CDC42•GppNHp was used as positive control. GST control experiments confirmed the specificity of the interaction between RHOG and IQGAP1. The upper part of the membrane was used for an anti-IQGAP1 immunoblotting, and the lower for an anti-GST. Densitometry analysis of relative IQGAP binding to GST-CDC42 or GST-RHOG (a. u., arbitrary unit) were performed in the next step. Bar charts at the right panel display the quantitation of detected signal in GST-pull down assay from a triplicate experiment.

Figure 2

RHO GTPases exhibit significantly different electrostatic properties. (A) The G domain organization of RAC1 indicates secondary structure elements, key functional regions and locations of residues crucial for IQGAP1 binding. (B) A multiple amino acid sequence alignment of canonical RHO GTPases revealed various residues outside of the switch regions that may determine their differential interactions with IQGAPs. IQGAP binders are colored green, and the nonbinders are colored red. (C) Structures in ribbon representation, solvent accessible proteins surfaces and electrostatic potential maps for RAC1 (PDB code, 1MH1), CDC42 (PDB code, 2QRZ), RHOA (PDB code, 1A2B), RND1 (PDB code, 2CLS), and RHOD (PDB code, 2J1L) are shown. Thr-25, Asn-26, Met-45, Asn-52, Gln-74, Val-85, and Ala-88 of RAC1 proposed to determine its specificity for the binding of IQGAPs are located on the surface, negatively charged residues on corresponding positions in, for example, RHOA and RND1 cause significant negative electrostatic potentials. Images were generated with the PyMOL molecular viewer. (D) The distribution of charged amino acids vary significantly among RHO GTPases despite their high sequence homology. Sequence alignment of the RHO GTPases used in this study reduced in a way that only loci containing at least one positively charged amino acid, i.e., arginine or lysine, or one negatively charged amino acid, i.e., glutamate or aspartate, were retain, respectively. It demonstrates diverse occurrence of charges in proteins molecules of RHO GTPases that is also reflected on huge differences of electrostatic potentials shown in C. They roughly also correspond to theoretical net charges for whole proteins that were obtained as sums of the +1 or −1 for positively or negatively charged residues, respectively. As only RHOoD and RIF were found to be electrically neutral while all other GTPases possess overall negative net charge, characteristic lobes of negative, red colored electrostatic potentials around the majority of proteins were observed (for reference see also Figure S5).

Figure 3

IQGAP1^C794 competes with DOCK2, p50^GAP, and PAK1 for binding RAC1. (A) The evaluated observed rate constants (k_obs), shown as bars, demonstrate that IQGAP1^C794 associates with RAC1 regardless of the presence of excess amounts of GDI1, TIAM1, TRIO, Plexin-B1, or p67^phox, while the association was blocked in the presence of DOCK2 or p50^GAP and completely abolished in the presence of PAK1. (B) The p50^GAP-stimulated GTPase activity of RAC1 was drastically reduced in the presence of IQGAP1^C794. (C) TIAM1- and DOCK2-catalyzed nucleotide exchange activity of RAC1 was not significantly changed in the presence of excess amounts of IQGAP1^C794. All measurements in (A–C), which are shown in detail in Figure S6, were obtained in triplicate. The data are expressed as the means ± S.D. (D) The left panel shows the structure of RAC1 (gray represents the surface) in complex with different RAC and CDC42 interacting partners (in different colored ribbons), including DOCK2^DHR2, TIAM1^DH-PH, TRIO^DH-PH, p50^GAP, GDI1, PAK1^GBD, p67^phoxTRP, and Plexin-B1^RBD. The right panel highlights the contact sites of these binding proteins on the surface of RAC1 in the corresponding colors. The protein database identification codes of the respective structures are indicated. (E) The complex structure of RAC3 (PDB code, 2IC5) and the CRIB motif of PAK1 (PDB code, 2QME) shows that T25, N26, M45, N52, and Q74 of RAC3 are in close proximity to the CRIB motif-binding region. Electrostatic potentials (right panel) show that the PAK1 CRIB motif generates an overall negative electrostatic surface potential.

Figure 4

Kinetic measurements of RAC1 and CDC42 variants binding IQGAP1^C794. The calculated association rates (k_on), dissociation rates (k_off), and dissociation constants (K_d) for the interaction of IQGAP1^C794 with different variants of RAC1 (A), CDC42 (B), and RHOA (C) are plotted as bar charts. All kinetic data are summarized in Table S1 and shown in Figure S7. The data are expressed as the means ± S.D.