Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.
Related Articles Millisecond Time-Resolved Solid-State NMR Reveals a Two-Stage Molecular Mechanism for Formation of Complexes between Calmodulin and a Target Peptide from Myosin Light Chain Kinase.
J Am Chem Soc. 2020 Dec 07;:
Authors: Jeon J, Yau WM, Tycko R
Abstract
Calmodulin (CaM) mediates a wide range of biological responses to changes in intracellular Ca2+ concentrations through its calcium-dependent binding affinities to numerous target proteins. Binding of two Ca2+ ions to each of the two four-helix-bundle domains of CaM results in major conformational changes that create a potential binding site for the CaM binding domain of a target protein, which also undergoes major conformational changes to form the complex with CaM. Details of the molecular mechanism of complex formation are not well established, despite numerous structural, spectroscopic, thermodynamic, and kinetic studies. Here, we report a study of the process by which the 26-residue peptide M13, which represents the CaM binding domain of skeletal muscle myosin light chain kinase, forms a complex with CaM in the presence of excess Ca2+ on the millisecond time scale. Our experiments use a combination of selective 13C labeling of CaM and M13, rapid mixing of CaM solutions with M13/Ca2+ solutions, rapid freeze-quenching of the mixed solutions, and low-temperature solid state nuclear magnetic resonance (ssNMR) enhanced by dynamic nuclear polarization. From measurements of the dependence of 2D 13C-13C ssNMR spectra on the time between mixing and freezing, we find that the N-terminal portion of M13 converts from a conformationally disordered state to an ?-helix and develops contacts with the C-terminal domain of CaM in about 2 ms. The C-terminal portion of M13 becomes ?-helical and develops contacts with the N-terminal domain of CaM more slowly, in about 8 ms. The level of structural order in the CaM/M13/Ca2+ complexes, indicated by 13C ssNMR line widths, continues to increase beyond 27 ms.
PMID: 33280387 [PubMed - as supplied by publisher]
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