title: Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix-helix interactions.
abstract: Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1-75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9 degrees C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., [Biochemistry 2002;41:15-20]), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1-74 to PCaM1-80). For the first 4, PCaM1-74 through PCaM1-77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5 degrees C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1-77 through PCaM1-80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of 15N-PCaM1-74 and 15N-PCaM1-80 and analysis of high-resolution structural models suggest these residues are latched to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains.
authors.affiliation: Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109, USA.
authors.forename: Laurel A
authors.initials: LA
authors.lastname: Faga
authors.forename: Brenda R
authors.initials: BR
authors.lastname: Sorensen
authors.forename: Wendy S
authors.initials: WS
authors.lastname: VanScyoc
authors.forename: Madeline A
authors.initials: MA
authors.lastname: Shea
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010247:Paramecium; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D002147:Calmodulin; D002118:Calcium