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Crystal structure and in vitro analysis of human IQGAP1 Calponin homology domain: implication for its interactions with Ca2+ bound Calmodulin and F‐actin
Author(s) -
Liu Jing,
Kurella Vinodh,
LeCour Louis F.,
Worthylake David Kent
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1013.1
Subject(s) - iqgap1 , calmodulin , dimer , isothermal titration calorimetry , calponin , actin , chemistry , binding site , actin cytoskeleton , cooperativity , plasma protein binding , biophysics , binding domain , cytoskeleton , crystallography , biochemistry , biology , cell , organic chemistry , enzyme
IQGAP1 is a 190kDa multi‐domain scaffold protein involved in cell signaling, cytoskeleton reorganization, cell‐cell adhesion via its dynamic interactions with various binding partners. A single copy Type‐3 Calponin homology domain locates at its amino terminus (residues 46–160). Here we have determined the crystal structure of human IQGAP1 residues 1–191 (CHD). In the crystal, two CHD molecules form a “head‐to‐head” homodimer. At high concentration in solution, the CHD can also form a dimer. Isothermal titration calorimetry (ITC) and F‐actin co‐sedimentation assays show that the CHD can bind to Ca2+/CaM and F‐actin, respectively. Ca2+/CaM does not affect the interaction between the CHD and F‐actin. The Calmodulin Target Database identified residues 91–103 of the CHD as a potential CaM binding site. Site‐directed mutagenesis and ITC indicate that 92LKKI95 is critical for Ca2+/CaM binding. Structure‐based sequence alignment predicts an F‐actin binding site locating in the dimer interface. A disulfide bond (K161C) was introduced to stabilize the dimer and mask potential F‐actin‐binding residues. Reduced F‐actin binding was observed upon dimer formation. Our results suggest that residues 92LKKI95 contribute to Ca2+/CaM binding, and that F‐actin binding utilizes residues located at the dimer interface. Our results also indicate the self‐association of full‐length IQGAP1 may be a parallel homodimer.