Calcium binding and conformational properties of calmodulin complexed With PEP-19

Abstract

PEP-19 is an IQ calmodulin (CaM) binding motif that inhibits apoptosis and protects cells against Ca2+ toxicity. We showed that PEP-19 interacts predominantly with the C-domain of CaM, and that it greatly increases the kon and koff rates of Ca2+ binding, but has little effect on KCa. Here we used solution NMR to characterize the calcium binding and conformational properties of the Ca2+-CaM-PEP-19 complex. Both 3JHNHA and 1H, 15N NOESY-HSQC experiments show the overall secondary structure of Ca2+-CaM is not greatly affected upon binding PEP-19. 15N backbone dynamics suggests that the Ca2+-CaM-PEP-19 complex shows large-scale dynamics. Most residues in the C domain of CaM that experience significant chemical exchange on μs to ms timescale form a hydrophobic patch to interact with PEP-19.We used a C-term fragment of CaM, CaM(76–148), which binds two Ca2+ ions, to determine the effect of PEP-19 on cooperative Ca2+ binding. Highly cooperative Ca2+ binding was seen in the absence of PEP-19, giving two sets of peaks in the 1H-15N HSQC spectra at substoichiometric levels of Ca2+, corresponding to apo and 2-Ca2+ bound forms of CaM(76–148). However, in the presence of PEP-19, cooperativity was largely lost and most residues in CaM(76–148) showed line broadening, and spitting into multiple peaks at low Ca2+ levels. Amide markers in the Ca2+ binding loops showed sequential Ca2+-binding first to site IV and then to site III. Furthermore, 1Hα, 13Cα chemical shift perturbations indicate that the β-strand in Ca2+ binding loop III shifts toward the random coil direction in the presence of PEP-19. This indicates that loss of cooperativity and increased in koff and kon rates induced by PEP-19 is caused by destabilizing the antiparallel β-sheet formed between Ca2+ binding sites III and IV in the C-domain of CaM.