Unraveling the molecular mechanism of interactions of the Rho GTPases Cdc42 and Rac1 with the scaffolding protein IQGAP2

Abstract

IQ motif-containing GTPase-activating proteins (IQGAPs) are scaffolding proteins playing central roles in cell-cell adhesion, polarity, and motility. The Rho GTPases Cdc42 and Rac1, in their GTP-bound active forms, interact with all three human IQGAPs. The IQGAP-Cdc42 interaction promotes metastasis by enhancing actin polymerization. However, despite their high sequence identity, Cdc42 and Rac1 differ in their interactions with IQGAP. Two Cdc42 molecules can bind to the Ex-domain and the RasGAP site of the GTPase-activating protein (GAP)-related domain (GRD) of IQGAP and promote IQGAP dimerization. Only one Rac1 molecule might bind to the RasGAP site of GRD and may not facilitate the dimerization, and the exact mechanism of Cdc42 and Rac1 binding to IQGAP is unclear. Using all-atom molecular dynamics simulations, site-directed mutagenesis, and Western blotting, we unraveled the detailed mechanisms of Cdc42 and Rac1 interactions with IQGAP2. We observed that Cdc42 binding to the Ex-domain of GRD of IQGAP2 (GRD2) releases the Ex-domain at the C-terminal region of GRD2, facilitating IQGAP2 dimerization. Cdc42 binding to the Ex-domain promoted allosteric changes in the RasGAP site, providing a binding site for the second Cdc42 in the RasGAP site. Of note, the Cdc42 "insert loop" was important for the interaction of the first Cdc42 with the Ex-domain. By contrast, differences in Rac1 insert-loop sequence and structure precluded its interaction with the Ex-domain. Rac1 could bind only to the RasGAP site of apo-GRD2 and could not facilitate IQGAP2 dimerization. Our detailed mechanistic insights help decipher how Cdc42 can stimulate actin polymerization in metastasis.

Keywords: CDC42; IQGAP2; Rac (Rac GTPase); Rac1; Ras; actin; actin polymerization; allosteric regulation; dimerization; molecular dynamics.

Figure 1.

A, crystal structures of Cdc42 (PDB entry 5CJP, chain A) and Rac1 (PDB entry 3TH5). The insert loops in an α-helical motif from both proteins are highlighted. In the insert loop structure and sequence, hydrophobic, polar/glycine, positively charged, and negatively charged residues are colored white (black), green, blue, and red, respectively. B, domain structure of IQGAP2 and crystal structure of GRD of IQGAP2 (GRD2) dimer with four Cdc42 molecules (PDB entry 5CJP). Ex-domains form a dimerization interface, and the C-term of the Ex-domain intertwines with the counterpart C-term of the other GRD2. Each monomeric unit of GRD2 contains two Cdc42 molecules at the RasGAP site and the Ex-domain. The number in parenthesis denotes two monomeric units of GRD2.

Figure 2.

A, RMSF plots of apo-GRD2 (left) and GRD2 from Ex-mode Cdc42-GRD2 complex (right). Green lines cover the RasGAP site residues, and the remaining residues (cyan lines) belong to the Ex-domain. In the right panel, the red ellipse represents the residues between 1124 and 1128 that conformationally change upon Ex-mode Cdc42 binding. In the right panel, C-term residues show higher fluctuation than 10 Å (up to 20 Å); however, to clearly observe and compare fluctuation profiles of GRD2s from different complexes, the y axis limit is determined as 10 Å. B, time series of snapshots of Ex-mode Cdc42-GRD2. Pink molecules are Cdc42s, and green molecules are GRD2, whereas cyan-highlighted residues are the C-terms of the Ex-domain of GRD2s.

Figure 3.

Allosteric pathways between Asn-132/Met-1124 (A), Asn-132/Thr-1125 (B), Asn-132/Ala-1126 (C), Asn-132/Gly-1127 (D), and Asn-132/Gly-1128 (E) of Ex-mode Cdc42 and GRD2 obtained by the WISP method. Pink structures are Cdc42s, and yellow structures are GRD2s. The red balls represent the residues involve in allosteric pathways, and the blue lines represent the possible paths followed by allosteric pathways.

Figure 4.

Interactions of Ex-mode Cdc42 (A) and Ex-mode Rac1 (B) with GRD2. On the right-hand side, stable salt bridges and stable H-bond formed between Ex-mode Cdc42 and GRD2 (A) and Ex-mode Rac1 and GRD2 (B) can be seen. Green molecules are GRD2, and pink/gray molecules are Cdc42/Rac1. Dark pink/dark gray regions are insert loops and switch I of Cdc42/Rac1. In the residue label, insert loop residues (†) and switch I residues (*) are indicated. Black dashes, salt bridges; orange dashes, H-bond. The residues Asn-132 and Lys-131 of Cdc42 are highlighted by boldface type.

Figure 5.

Binding free energies of Ex-mode Cdc42-GRD2, Ex-mode Rac1-GRD2, RasGAP mode Cdc42-GRD2, and RasGAP mode Rac1-GRD2 in kcal/mol calculated by the MM-GBSA method. In the calculation, gas phase contribution, 〈ΔG_gas〉, the solvation energy contribution, 〈ΔG_sol〉, and the entropic contribution, −TΔS, combine to give the average binding free energy, 〈ΔG_b〉. In the box graphs, the red dot and red line denote the mean and median values, respectively. Whiskers above and below the box denote the 90th and 10th percentiles.

Figure 6.

A, RMSF plots of apo-GRD2 (left) and GRD2 from Ex-mode Rac1-GRD2 complex (right). Green lines cover the RasGAP site residues, and the remaining residues (cyan lines) belong to the Ex-domain. B, time series of snapshots of Ex-mode Rac1-GRD2. Gray molecules are Rac1, and green molecules are GRD2, whereas cyan-highlighted residues are the C-terms of the Ex-domain of GRD2s.

Figure 7.

Interactions of RasGAP mode Cdc42 (A) and RasGAP mode Rac1 (B) with GRD2. On the right-hand side, stable salt bridges formed between RasGAP mode Cdc42 and GRD2 (A) and RasGAP mode Rac1 and GRD2 (B) can be seen. Green molecules are GRD2, and pink/gray molecules are Cdc42/Rac1. Dark pink/dark gray regions are switch I and switch II of Cdc42/Rac1. In the residue label, switch I residues (*) and switch II residues (**) are indicated. Dashed lines, salt bridges.

Figure 8.

Time series of snapshots of RasGAP mode Cdc42-GRD2 (A) and RasGAP mode Rac1-GRD2 (B). Pink molecules are Cdc42, gray molecules are Rac1, and green molecules are GRD2, whereas cyan-highlighted residues are the C-terms of the Ex-domain of GRD2s.

Figure 9.

A, binding free energies of RasGAP mode Cdc42-GRD2^Y1106A and RasGAP mode Rac1-GRD2^Y1106A in kcal/mol calculated by the MM-GBSA method. B and C, stable salt bridges formed between RasGAP mode Cdc42 and GRD2^Y1106A (B) and RasGAP mode Rac1 and GRD2^Y1106A (C). Green molecules are GRD2, and pink/gray molecules are Cdc42/Rac1. In the residue label, switch I residues (*) and switch II residues (**) are indicated. Dashed lines, salt bridges. The mutated Ala-1106 residue is highlighted by boldface type.

Figure 10.

Mutation of Tyr-1193 of IQGAP1 impairs binding to Cdc42 and Rac1. 2 μg of purified His-Cdc42-Q61L (A) or His-Rac1-Q61L (B) was incubated with 3 μg of GST-IQGAP1-C (wildtype (WT) or Y1193A (1193)) on glutathione-Sepharose beads. Incubation with GST alone was used as a negative control. After incubation, samples were washed and resolved by SDS-PAGE. The top half of the gel (containing the GST-IQGAP1 constructs) was stained with Coomassie Blue. The bottom half of the gel, which contains Cdc42 or Rac1, was transferred to PVDF and probed with anti-His antibodies. Blots were imaged with Image Studio version 2.0 (LI-COR Biosciences). His-tagged Cdc42 or Rac1 that was not incubated with GST proteins was processed by SDS-PAGE and Western blotting (Input). The positions of migration of molecular weight markers are depicted. Data are representative of at least four independent experiments.

Figure 11.

Interaction of GRD2 with Cdc42 (A) and Rac1 (B and C). A, schematic diagram illustrating the release of C-term and allosteric change in RasGAP site upon Ex-mode Cdc42 binding. Allosteric change in the RasGAP site induces binding of the second Cdc42 to RasGAP site, and GRD2 with two Cdc42s can dimerize. B, Rac1 cannot bind to the Ex-domain and cannot induce allosteric change in the RasGAP site and release of C-term. C, Rac1 can bind to the RasGAP site of apo-GRD2 but cannot induce the release of C-term. Therefore, Rac1 cannot facilitate GRD2 dimerization.