Conformational Selection and Functional Dynamics of Calmodulin: A ¹⁹F Nuclear Magnetic Resonance Study

Abstract

Calcium-bound calmodulin (CaM-4Ca(2+)) is innately promiscuous with regard to its protein interaction network within the cell. A key facet of the interaction process involves conformational selection. In the absence of a binding peptide, CaM-4Ca(2+) adopts an equilibrium between a native state (N) and a weakly populated near-native peptide-bound-like state (I), whose lifetime is on the order of 1.5 ms at 37 °C, based on (19)F nuclear magnetic resonance (NMR) Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion measurements. This peptide-bound-like state of CaM-4Ca(2+) is entropically stabilized (ΔS = 280 ± 35 J mol(-1) K(-1)) relative to the native state, water-depleted, and likely parental to specific bound states. Solvent depletion, conformational selection, and flexibility of the peptide-bound-like state may be important in priming the protein for binding. At higher temperatures, the exchange rate, kex, appears to markedly slow, suggesting the onset of misfolded or off-pathway states, which retards interconversion between N and I. (19)F NMR CPMG relaxation dispersion experiments with both CaM-4Ca(2+) and the separate N-terminal and C-terminal domains reveal the cooperative role of the two domains in the binding process and the flexibility of the N-terminal domain in facilitating binding. Thus, when calcium binds, calmodulin establishes its interaction with a multitude of protein binding partners, through a combination of conformational selection to a state that is parental to the peptide-bound state and, finally, induced fit.

Effect of Ca2+on the promiscuous target-protein binding mechanism of calmodulin

“…We hypothesize that the longranged electrostatic interactions of the N-CaM charged residues may initiate fast binding while the hydrophobic pocket in C-CaM may account for selectivity. Previously published studies using NMR [23] proposed that C-CaM is selective while N-CaM binds afterwards through induced fit mechanism, or a coupled conformational selection mechanism initiated by C-CaM [60]. This is in line with the argument given here on C-CaM selectivity and N-CaM flexibility.…”

“…Both CaM dynamics and motif-dependent target-protein binding have been extensively studied via both experiments [2,8,[10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] and simulations. Indeed, all-atom molecular dynamics (MD) simulations allow for a detailed description with molecular insights.…”

Probing protein flexibility reveals a mechanism for selective promiscuity

“…Generally speaking, one assumes a conformational selection scenario if the apo protein ensemble samples bound-like states (apo BL ) (Boehr et al, 2009; Hoang and Prosser, 2014). If not, one assumes induced fit (Schon et al, 2002).…”

Determination of the binding mechanism of histone deacetylase inhibitors

“…Compared with this, the IF model consists of an initial ligand binding step to the target protein followed by a mutual conformational adaptation of the protein and the ligand to achieve optimal steric and electrostatic complementarity. In reality, both general types of interaction may coexist in many documented cases (Agafonov, Wilson, Otten, Buosi, & Kern, ; Dogan, Gianni, & Jemth, ; Faleev et al., ; Hoang & Prosser, ). If experimental methods are sufficiently accurate, even more complex multistep binding mechanism has been reported (Dogan, Schmidt, Mu, Engstrom, & Jemth, ; Huddleston, Schroeder, Johnson, & Whitman, ; Meyners & Meyer‐Almes, ; c; Sykora & Meyer‐Almes, ).…”