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

Abstract

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/4Ca2+ 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.

Combined Pulsed Electron Double Resonance EPR and Molecular Dynamics Investigations of Calmodulin Suggest Effects of Crowding Agents on Protein Structures

“…Very recently, a study by the Clore group investigated the time-resolved binding of CaM to a peptide derived from myosin light-chain kinase using DEER. Although also not directly comparable due to labeling sites used (S17C/A128C), the final peptide-bound state they measured is broadly consistent with our observations . Another study by the Goldfarb group compared DEER and NMR measurements of CaM binding to two peptides (IQ and MARCKS), representing two different binding conformations of CaM, both of which have corresponding crystal structures of CaM bound to the respective peptide .…”

“…Although also not directly comparable due to labeling sites used (S17C/A128C), the final peptide-bound state they measured is broadly consistent with our observations. 49 Another study by the Goldfarb group compared DEER and NMR measurements of CaM binding to two peptides (IQ and MARCKS), representing two different binding conformations of CaM, both of which have corresponding crystal structures of CaM bound to the respective peptide. 50 It was found that solution measurements of CaM bound to the IQ peptide were consistent with the crystal structure, but with the MARCKS peptide, there were significant discrepancies.…”

Quantitative Agreement between Conformational Substates of Holo Calcium-Loaded Calmodulin Detected by Double Electron–Electron Resonance EPR and Predicted by Molecular Dynamics Simulations

“…33,34 on holo CaM/4Ca 2+ and of the compact crystal structure (1PRW) 19 of holo CaM/4Ca 2+ together with the corresponding P(r) distributions between A17C-R1 and A128C-R1 calculated from a R1 rotamer library. 18 The MD clusters can be grouped into six families (A−F), with the compact crystal structure as a seventh family (G). The positions of the R1 nitroxide labels are indicated by red spheres located at the relevant backbone Cα atoms for visualization purposes only.…”

“…The P(r) distributions are calculated from a weighted ensemble of nitroxide oxygen positions based on molecular coordinates of the structures, as described previously. 18 Because the nitroxide labels are represented as a weighted ensemble, the shape of the calculated P(r) distribution can be non-Gaussian and can include shoulders or separate peaks that correspond to distinct regions of conformational space occupied by one or both nitroxide labels. clustering, based on Gaussian mixtures, was used to extract 16 well-defined core substates (clusters) 33 from the free energy landscape of a 460 ns replica-exchange solute-tempering MD simulation on holo CaM/4Ca 2+ .…”

“…DEER yields probability distance distributions, P(r), between pairs of spin labels, 35 and the two sites of R1 nitroxide spin-labeling 36,37 intermediate (interspin distance ∼ 21 Å) or the final complex (interspin distances ∼21 and 26 Å arising from two distinct regions of conformational space occupied by the R1 label at A128C). 18 DEER Data Acquisition. The experimental DEER data were acquired at Q-band and 50 K on CaM/4Ca 2+ (A17C-R1/ A128C-R1).…”