Ca2+-Dependent Switch of Calmodulin Interaction Mode with Tandem IQ Motifs in the Scaffolding Protein IQGAP1

Abstract

IQ domain GTPase-activating scaffolding protein 1 (IQGAP1) mediates cytoskeleton, cell migration, proliferation, and apoptosis events. Calmodulin (CaM) modulates IQGAP1 functions by binding to its four tandem IQ motifs. Exactly how CaM binds the IQ motifs and which functions of IQGAP1 CaM regulates and how are fundamental mechanistic questions. We combine experimental pull-down assays, mutational data, and molecular dynamics simulations to understand the IQ-CaM complexes with and without Ca²⁺ at the atomic level. Apo-CaM favors the IQ3 and IQ4 motifs but not the IQ1 and IQ2 motifs that lack two hydrophobic residues for interactions with apo-CaM's hydrophobic pocket. Ca²⁺-CaM binds all four IQ motifs, with both N- and C-lobes tightly wrapped around each motif. Ca²⁺ promotes IQ-CaM interactions and increases the amount of IQGAP1-loaded CaM for IQGAP1-mediated signaling. Collectively, we describe IQ-CaM binding in atomistic detail and feature the emergence of Ca²⁺ as a key modulator of the CaM-IQGAP1 interactions.

Figure 1.

Mutations of IQ3 and IQ4 motifs alter calmodulin binding. In vitro binding analysis of IQ domain constructs of IQGAP1. (A) The wild-type (WT) IQ domain (amino acids 717–916), (B) IQ3R, and (C) IQ4R were labeled with [³⁵S]Met using T_NT. Samples were incubated with purified recombinant GST-CaM constructs, namely, full-length (F, amino acids 1–148), N-half (N, amino acids 1–74), and C-half (C, amino acids 75–148) or GST alone. In the top panels, the bound proteins were resolved by SDS–PAGE and imaged by autoradiography. Input (Inp.) is 10% of the IQ domain used for the binding assay. Representative images of three independent experiments are shown. Autoradiographs were quantified with Li-Cor Image Studio Software. The amount of binding of each IQ domain was normalized to CaM-F in Ca²⁺. The graphs depict binding to CaM-F (red), CaM-N (blue), CaM-C (green), or GST (white). Data are means ± the standard deviation (n = 3). Significance was determined by ANOVA: *p < 0.05; **p < 0.005; ***p < 0.0005; ns, not significant.

Figure 2.

Characterization of IQ1–IQ4 motifs in IQGAP1. The structures are obtained from homology modeling. The hydrophobic, hydrophilic, basic, and acidic residues are colored white, green, blue, and red, respectively. Asterisks denote the conserved residues in IQ core sequences.

Figure 3.

Properties of IQ motifs binding to apo-CaM. (A) RMSF values of apo-CaM and (B) interface areas between IQ motifs and CaM in the apo-CaM–IQ complexes. (C and D) Comparison of the sequences and structures of modeled apo-CaM–IQ complexes, with a cartoon depicting the apo-CaM–IQ interaction modes. Color code: red for CaM, pink for the IQ1 motif, yellow for the IQ2 motif, cyan for the IQ3 motif, and purple for the IQ4 motif. The rectangular box in panel C highlights two residues of IQ1–IQ4 in the hydrophobic pockets of apo-CaMs. The double-ended arrows in the middle panels of panel D indicate the motion of the N-lobe of apo-CaMs. Important residue contacts in the apo-CaM–IQ3 and apo-CaM–IQ4 complexes are highlighted.

Figure 4.

Properties of IQ motifs binding to Ca²⁺-CaM. Snapshots of Ca²⁺-CaM interacting with (A) IQ1, (B) IQ2, (C) IQ3, and (D) IQ4 motifs, displayed by a cartoon depicting the Ca²⁺-CaM–IQ interaction mode. (E) RMSF values of Ca²⁺-CaM and (F) interface areas between IQ motifs and Ca²⁺-CaM in the Ca²⁺-CaM–IQ complexes. The figure shows that the conserved basic and hydrophobic residues, especially the “I” of IQ (Leu⁷⁵² for IQ1, Ile⁷⁸² for IQ2, Ile⁸¹² for IQ3, and Ile⁸⁴² for IQ4), exhibit significant Ca²⁺-CaM–IQ interactions. The blue dots denote the Ca²⁺ in the complexes.

Figure 5.

Schematic illustration of Ca²⁺-dependent CaM–IQ interactions in IQGAP1.