IQGAP1 is a multidomain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) that contains the entire calponin homology domain. The structure of the CHDF is very similar to those of other type 3 calponin homology domains like those from calponin, Vav, and the yeast IQGAP1 ortholog Rng2. However, in the crystal, two CHDF molecules form a "head-to-head" or parallel dimer through mostly hydrophobic interactions. Binding experiments indicate that the CHDF binds to both F-actin and Ca²⁺/calmodulin, but binding is mutually exclusive. On the basis of the structure, two dimer interface substitutions were introduced. While CHDFL157D disrupts the dimer in gel filtration experiments, oxidized CHDFK161C stabilizes the dimer. These results imply that the CHDF forms the same dimer in solution that is seen in the crystal structure. The disulfide-stabilized dimer displays a reduced level of F-actin binding in sedimentation assays and shows no binding to Ca²⁺/calmodulin in isothermal titration calorimetry (ITC) experiments, indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues ⁹³KK⁹⁴ are important for CaM binding, and indeed, the ⁹³EE⁹⁴ double mutation displays a reduced level of binding to Ca²⁺/calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca²⁺/calmodulin binding, and the ⁹³KK⁹⁴ pair, near the interface, is also used for Ca²⁺/calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer.

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