The IQGAP1 N-terminus Forms Dimers and the Dimer Interface is Required for Binding F-actin and Ca 2+ /calmodulin

, Abstract IQGAP1 is a multi-domain scaffold protein involved in many cellular processes. We have determined the crystal structure of an N-terminal fragment spanning residues 1-191 (CHDF hereafter) which contains the entire calponin homology domain. The structure of the CHDF is very similar to 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 Ca 2+ /calmodulin, but binding is mutually exclusive. Based on 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 reduced F-actin binding in sedimentation assays, and shows no binding to Ca 2+ /calmodulin in isothermal titration calorimetry (ITC) experiments indicating that interface residues are utilized for both binding events. The Calmodulin Target Database predicts that residues 93 KK 94 are important for CaM binding, and indeed the 93 EE 94 double mutation displays reduced binding to Ca 2+ /calmodulin in ITC experiments. Our results indicate that the CHDF dimer interface is used for both F-actin and Ca 2+ /calmodulin binding, and 93 KK 94 , near the interface, are also used for Ca 2+ /calmodulin binding. These results are also consistent with full-length IQGAP1 forming a parallel homodimer. and the dimer is mainly stabilized through hydrophobic interactions across a shape-complementary interface. We find that the CHDF can form a dimer in solution, and site-directed mutagenesis studies based on the dimer seen in the crystal indicate that the solution dimer is very similar to the crystal dimer. Our results, using a fragment representing only ~ 12% of the intact protein, suggests that full-length IQGAP1 forms parallel dimers in vivo and in doing so, juxtaposes its common domains and motifs. A structure-based sequence alignment reveals a potential CH1-like ABS2 which in the structure exists at the CHDF dimer interface. A dimer interface substitution K161C, when oxidized, shows significantly reduced binding to F-actin suggesting that this ABS2 is required for F-actin binding. Our ITC data modeling indicates that the wild-type CHDF binds to Ca 2+ /CaM in a “two-site sequential” mode. Oxidized CHDFK161C shows a complete loss of binding to Ca 2+ /CaM indicating that complete dissociation of the CHDF dimer is required for CaM binding. The Calmodulin Target Database predicts a CaM binding site within CHDF residues 91-103 which do not lie at the dimer interface. Two consecutive lysines within this putative CaM-binding site are critical to the Database prediction, and the 93 KK 94 → 93 EE 94 double mutation in the CHDF results in a significant reduction in binding to Ca 2+ /CaM. In conclusion, our results suggest that IQGAP1 forms a parallel dimer, and the N-termini of the dimer, containing the CH domain, must dissociate to allow Ca 2+ /CaM or F-actin binding.