Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca ²⁺ -ligated calmodulin.

Publisher: National Academy of Sciences Country of Publication: United States NLM ID: 7505876 Publication Model: Print Cited Medium: Internet ISSN: 1091-6490 (Electronic) Linking ISSN: 00278424 NLM ISO Abbreviation: Proc Natl Acad Sci U S A Subsets: MEDLINE

Original Publication: Washington, DC : National Academy of Sciences

Calcium/*chemistry ; Calmodulin/*chemistry; Calcium/metabolism ; Calmodulin/metabolism ; Electron Spin Resonance Spectroscopy ; Humans ; Kinetics ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Domains ; Protein Folding

Recent advances in rapid mixing and freeze quenching have opened the path for time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state NMR of protein-substrate interactions. DEER, in conjunction with phase memory time filtering to quantitatively extract species populations, permits monitoring time-dependent probability distance distributions between pairs of spin labels, while solid-state NMR provides quantitative residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein. Here, we demonstrate the power of these combined approaches to unravel the kinetic and structural pathways in the binding of the intrinsically disordered peptide substrate (M13) derived from myosin light-chain kinase to the universal eukaryotic calcium regulator, calmodulin. Global kinetic analysis of the data reveals coupled folding and binding of the peptide associated with large spatial rearrangements of the two domains of calmodulin. The initial binding events involve a bifurcating pathway in which the M13 peptide associates via either its N- or C-terminal regions with the C- or N-terminal domains, respectively, of calmodulin/4Ca ²⁺ to yield two extended "encounter" complexes, states A and A*, without conformational ordering of M13. State A is immediately converted to the final compact complex, state C, on a timescale τ ≤ 600 μs. State A*, however, only reaches the final complex via a collapsed intermediate B ( τ ∼ 1.5 to 2.5 ms), in which the peptide is only partially ordered and not all intermolecular contacts are formed. State B then undergoes a relatively slow ( τ ∼ 7 to 18 ms) conformational rearrangement to state C.
Competing Interests: The authors declare no competing interest.
(Copyright © 2022 the Author(s). Published by PNAS.)

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Z01 DK029023 United States ImNIH Intramural NIH HHS; Z01 DK029029 United States ImNIH Intramural NIH HHS

Keywords: conformational transitions; coupled folding and binding; rapid freeze quenching; substrate binding pathways

0 (Calmodulin)
SY7Q814VUP (Calcium)

Date Created: 20220202 Date Completed: 20220307 Latest Revision: 20221005

20231215

PMC8833187

10.1073/pnas.2122308119

35105816