Basic interdomain boundary residues in calmodulin decrease calcium affinity of sites I and II by stabilizing helix–helix interactions

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°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°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. Proteins 2003;50:381–391. © 2003 Wiley-Liss, Inc.