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

Abstract

Calcium-loaded calmodulin (CaM/4Ca 2+ ) comprises two domains that undergo rigid body reorientation from a predominantly extended conformation to a compact one upon binding target peptides. A recent replica-exchange molecular dynamics (MD) simulation on holo CaM/4Ca 2+ suggested the existence of distinct structural clusters (substates) along the path from extended to compact conformers in the absence of substrates. Here, we experimentally demonstrate the existence of CaM/4Ca 2+ substates trapped in local minima by three freezing/annealing regimes (slow, 40 s; intermediate, 1.5 s; fast, 0.5 ms) using pulsed Q-band double electron−electron resonance (DEER) EPR spectroscopy to measure interdomain distances between nitroxide spinlabels positioned at A17C and A128C in the N-and C-terminal domains, respectively. The DEER echo curves were directly fit to population-optimized P(r) pairwise distance distributions calculated from the coordinates of the MD clusters and compact crystal structure. DEER data on fully deuterated CaM/4Ca 2+ were acquired at multiple values of the second echo period (10−35 μs) and analyzed globally to eliminate instrumental and overfitting artifacts and ensure accurate populations, peak positions, and widths. The DEER data for all three freezing regimes are quantitatively accounted for within experimental error by 5−6 distinct conformers comprising a predominantly populated extended form (60−75%) and progressively more compact states whose populations decrease as the degree of compactness increases. The shortest interdomain separation is found in the compact crystal structure, which has an occupancy of 4−6%. Thus, CaM/4Ca 2+ samples high energy local minima comprising a few discrete substates of increasing compactness in a rugged energy landscape.

chiLife: An open-source Python package for in silico spin labeling and integrative protein modeling

“…These distance distributions directly provide information on protein tertiary and quaternary structure. Coupled with high-resolution structural techniques, SDSL EPR provides insight into protein conformational landscapes and how they change in response to different stimuli [16][17][18][19][20][21][22][23][24][25][26][27]. Accordingly, SDSL EPR is particularly useful for validation and refinement of protein structural models as well as expansion of these models to include conformational heterogeneity and distinct alternate conformational states.…”

Direct observation of negative cooperativity in a detoxification enzyme at the atomic level by Electron Paramagnetic Resonance spectroscopy and simulation

“…Here, we characterize the ligand‐free conformational ensemble of the hGSTA1‐1 enzyme in atomistic detail using a combination of pulsed EPR distance measurements and weighted ensemble (WE) molecular dynamics (MD) simulations. Pulsed EPR distance measurements (Bonora et al, 2004; Borbat & Freed, 1999; Jeschke et al, 2000; Kulik et al, 2001; Milikisyants et al, 2009; Milov et al, 1998; Pannier et al, 2011), generally enabled by site‐directed spin labeling, are powerful probes for protein–protein (Nyenhuis et al, 2020; Park et al, 2006; Schmidt et al, 2019) and protein–nucleic acid (Krumkacheva et al, 2019; Stone et al, 2008) interactions, metal and ligand binding sites (Abdullin et al, 2015; Gamble Jarvi et al, 2019; Yin et al, 2017), and macromolecular conformational changes (Altenbach et al, 2008; Barth et al, 2018; Dalmas et al, 2014; Dastvan et al, 2019; Evans et al, 2016; Evans et al, 2020; Hett et al, 2021; Kear et al, 2009; Schmidt et al, 2022; Stewart et al, 2022). Such measurements can also be performed in cells (Igarashi et al, 2010; Joseph et al, 2015; Martorana et al, 2014; Meichsner et al, 2021; Roser et al, 2016; Teucher et al, 2019).…”

“…protein-protein (Nyenhuis et al, 2020;Park et al, 2006;Schmidt et al, 2019) and protein-nucleic acid (Krumkacheva et al, 2019;Stone et al, 2008) interactions, metal and ligand binding sites (Abdullin et al, 2015;Gamble Jarvi et al, 2019;Yin et al, 2017), and macromolecular conformational changes (Altenbach et al, 2008;Barth et al, 2018;Dalmas et al, 2014;Dastvan et al, 2019;Evans et al, 2016;Evans et al, 2020;Hett et al, 2021;Kear et al, 2009;Schmidt et al, 2022;Stewart et al, 2022). Such measurements can also be performed in cells (Igarashi et al, 2010;Joseph et al, 2015;Martorana et al, 2014;Meichsner et al, 2021;Roser et al, 2016;Teucher et al, 2019).…”

Structural Insights into the Binding and Degradation Mechanisms of Protoporphyrin IX by the Translocator Protein TSPO

“…We evaluated the catalytic degradation reaction of PpIX mediated by both wild-type (WT) Bc TSPO and Bc TSPO variants carrying mutations at key residues. Moreover, we perform the pulsed double electron–electron resonance (DEER) − technique to measure the distances between selected spin-labeled pairs in Bc TSPO embedded in nanodiscs. Hereafter, we refer to Bc TSPO as TSPO for simplicity.…”