An EF-hand phage display study of calmodulin subdomain pairing

The interaction between the two EF-hands, EF3 and EF4, in the C-terminal do main of vertebrate calmodulin is addressed using an EF-hand phage display l ibrary. Significant specificity is observed in the presence of Ca2+, as EF3 -EF4 heterodimers are favored over EF3-EF3 and EF4-EF4 homodimers. Primaril y EF4-type (and not EF3-type) amino acids are selected when an EF3 peptide is used as the target and nice versa. The results show that this specificit y is promoted by several factors. There are three positions, corresponding to Phe89, Ala102, and Leu105, that are strongly selected as EF3-type hydrop hobic residues with an EF4 target. When EF3 is the target peptide, EF4-type residues, Ile125, Tyr138 and Phe141, are selected. Remarkably, this subset consists of the same three residue positions in EF3 or EF4 and seems to be involved in specifying the heterodimer preference in both cases. Ln additi on electrostatic repulsion between the acidic monomers in an EF4 homodimer may further influence the preferred stability of heterodimers. This hypothe sis is based on Me observation that positively charged residues are strongl y selected at four positions when EF4 is the target. A survey of EF-hand pa irs suggests that charge separation is a common way to achieve efficient at traction of Ca2+ without causing electrostatic repulsion between the subdom ains. No significant specificity of binding is observed in the ion free sta te or in the presence of magnesium as no sequence is preferentially selecte d. The residues at the interface between the two EF-hands are thus highly o ptimized for the Ca2+ bound state. At some residue positions, EF3-type amin o acids are chosen with EF3-target in the presence of Ca2+. These residues are not involved in the preference for heterodimer over homodimer formation , but represent key positions to mutate in the intact domain to stabilize i ts Ca2+-bound state. (C) 2000 Academic Press.