The IQ domains in neuromodulin and PEP19 represent two major functional classes

The Ca2+-binding protein calmodulin (CaM1) participates at multiple levels in essentially all cellular processes. It interacts with an estimated 100 intracellular proteins, whose activities it modulates to varying degrees with varying dependencies on the intracellular free Ca2+ concentration. CaM contains two pairs of EF hand Ca2+-binding domains, each comprising a globular structure which we term a “lobe”. The two lobes are joined by a solvent-exposed flexible helix (1). Due to cooperativity within each pair of Ca2+-binding sites (Ca2+)2CaM, with either the N-ter or C-ter EF hand pair occupied, and (Ca2+)4CaM are the major Ca2+ liganded species produced (2). A number of proteins, including neuromodulin, neurogranin and PEP19, the unconventional myosins, ion channels (3–5), and modulators of small G-proteins, bind CaM through so called “IQ domains”. Depending upon their amino acid sequences, these domains can bind Ca2+-free and Ca2+-bound forms of CaM with quite different degrees of preference, allowing for a variety of Ca2+-dependent switching behaviors (4). IQ domains were first identified as light chain subunit binding sites in conventional myosins, and the consensus sequence was defined as IQxxxRGxxxR (4, 6). As additional homologous domains have been discovered in other proteins a less restricted consensus sequence has emerged: [I,L,V]QxxxR[G,x]xxx[R,K] (4, 6). Structural studies demonstrate that a narrow hydrophobic cleft in the Ca2+-free C-ter CaM lobe and in the homologous regions in the myosin light chain subunits is involved in recognition of the “IQ” amino acid pair in the IQ domain (7–9). The Ca2+-free N-ter lobes in CaM and the light chains contain no such cleft, and their interactions with the IQ domain appear to be variable (7–9). Structural and biophysical studies indicate that in the absence of Ca2+ there are two types of light chain or CaM complexes with IQ domains: a compact structure in which the N-ter lobe interacts closely with the IQ domain, and an extended form in which the N-ter lobe is only weakly associated (7, 8). The compact structure is formed if the semi-conserved central Gly residue in the IQ consensus sequence is present, and the extended structure is formed if there is a bulky amino acid at this position (7, 8). Extensive hydrophobic surfaces are exposed on both CaM lobes when they are replete with Ca2+, and these appear to enfold IQ domains to form structures similar to those observed with other types of Ca2+-saturated CaM complexes (10, 11). Structural and biophysical studies further suggest that IQ domains adopt an α-helical conformations when they are bound to Ca2+-free or Ca2+-saturated CaM (7–11). The semi-conserved Gly in the IQ domain consensus sequence is present in neuromodulin, but has been replaced by a Lys in PEP19. The complexes between CaM and the IQ domains in these proteins are therefore expected to belong to different structural classes. In order to investigate the possible functional correlates of these classes, we compared the affinities of the Ca2+-free, Ca2+-saturated and intermediate Ca2+-bound CaM complexes with the neuromodulin and PEP19 IQ domains. Our results suggest that the IQ domains in neuromodulin and PEP19 represent two major functional classes whose complexes with CaM each exhibit a distinctive pattern of Ca2+-dependent stability changes.