DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution

• Published in: Structure (London) 2022-06, Vol.30 (6), p.813-827.e5
• Main Authors: Jash, Chandrima, Feintuch, Akiva, Nudelman, Shira, Manukovsky, Nurit, Abdelkader, Elwy H., Bhattacharya, Sudeshna, Jeschke, Gunnar, Otting, Gottfried, Goldfarb, Daniella
• Format: Article
• Language: English
• Subjects: calmodulin; DEER; EPR; IQ-peptide; lanthanoid ions; MARCKS; NMR; protein-peptide interaction
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2022.03.005

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure. [Display omitted] •The target peptides MARCKS and IQ bind CaM both in its apo- and holo-states•In solution, the binding of MARCKS to holo-CAM does not generate a closed conformation•In solution, holo-CaM with bound IQ has a closed conformation, albeit heterogeneous•Gd(III)/nitroxide DEER distance restraints give a solution holo-CaM/IQ structural model Jash et al. track the conformational changes calmodulin undergoes on binding MARCKS and IQ peptides in solution and report significant differences in their binding modes. This is achieved by various Gd(III) and nitroxide spin labeling schemes for CaM and the peptides combined with EPR inter-spin distance and paramagnetic NMR measurements.