Atomic resolution experimental phase information reveals extensive disorder and bound 2-methyl-2,4-pentanediol in Ca²⁺-calmodulin

Abstract

Calmodulin (CaM) is the primary calcium signaling protein in eukaryotes and has been extensively studied using various biophysical techniques. Prior crystal structures have noted the presence of ambiguous electron density in both hydrophobic binding pockets of Ca2+-CaM, but no assignment of these features has been made. In addition, Ca2+-CaM samples many conformational substates in the crystal and accurately modeling the full range of this functionally important disorder is challenging. In order to characterize these features in a minimally biased manner, a 1.0 Å resolution single-wavelength anomalous diffraction data set was measured for selenomethionine-substituted Ca2+-CaM. Density-modified electron-density maps enabled the accurate assignment of Ca2+-CaM main-chain and side-chain disorder. These experimental maps also substantiate complex disorder models that were automatically built using low-contour features of model-phased electron density. Furthermore, experimental electron-density maps reveal that 2-methyl-2,4-pentanediol (MPD) is present in the C-terminal domain, mediates a lattice contact between N-terminal domains and may occupy the N-terminal binding pocket. The majority of the crystal structures of target-free Ca2+-CaM have been derived from crystals grown using MPD as a precipitant, and thus MPD is likely to be bound in functionally critical regions of Ca2+-CaM in most of these structures. The adventitious binding of MPD helps to explain differences between the Ca2+-CaM crystal and solution structures and is likely to favor more open conformations of the EF-hands in the crystal.

Crystal structure of Arabidopsis thaliana calmodulin7 and insight into its mode of DNA binding

“…Solvent assessable area was calculated using the pisa server and it was found that AtCaM7 structure has the highest solvent assessable area of 9868.5 Å 2 in comparison with other CaM, mentioned above. Recently it has been shown that MPD molecule is likely to favor more open conformations of the EF‐hands in the crystal of CaM . This could be the reason of having high solvent‐assessable area in case of AtCaM7.…”

DelPhiForce web server: electrostatic forces and energy calculations and visualization

“…It can be seen that the total electrostatic force drives the ectodomain toward its binding partner indicating the dominant role of electrostatic forces in ectodomain homo-dimerization. Domain-domain interactions: this case illustrates the interdomain interactions of calmodulin in calcium loaded and peptide free state (PDB: 5E1K) (Lin et al, 2016). The total electrostatic force keeps the two lobes of calmodulin apart, thus helping it to retain the open conformational state of calcium loaded calmodulin (Fig.…”

A Free-Energy Landscape Analysis of Calmodulin Obtained from an NMR Data-Utilized Multi-Scale Divide-and-Conquer Molecular Dynamics Simulation

“…Monomeric structures of both Ca 2+ -bound CaM (holo-CaM) [28][29][30][31][32][33][34][35][36][37] and unbound CaM (apo-CaM) [38,39] have been studied experimentally. For example, X-ray crystallography and nuclear magnetic resonance (NMR) experiments have shown that CaM has two almost symmetrical globular domains; the domains are separated by a long linker region.…”

“…The linker region is disordered and flexibly connects the N-lobe and C-lobe, and then, the N-and C-lobe are expected to almost freely move independently from each other in the solution [38,39]. On the other hand, when Ca 2+ binds, the EFloop is pinched by Ca 2+ and the interaction between the entering-and exiting-helix of each EF-hand is inhibited, causing the helices to separate each other [28,29,[31][32][33][34][35][36][37]. This structural change exposes hydrophobic amino acids buried in the helix bundle, forming a hydrophobic pocket; the pocket is the binding site for the target protein [19,20].…”