Nucleotide-dependent conformational states of actin

Abstract

The influence of the state of the bound nucleotide (ATP, ADP-Pi, or ADP) on the conformational free-energy landscape of actin is investigated. Nucleotide-dependent folding of the DNase-I binding (DB) loop in monomeric actin and the actin trimer is carried out using all-atom molecular dynamics (MD) calculations accelerated with a multiscale implementation of the metadynamics algorithm. Additionally, an investigation of the opening and closing of the actin nucleotide binding cleft is performed. Nucleotide-dependent free-energy profiles for all of these conformational changes are calculated within the framework of metadynamics. We find that in ADP-bound monomer, the folded and unfolded states of the DB loop have similar relative free-energy. This result helps explain the experimental difficulty in obtaining an ordered crystal structure for this region of monomeric actin. However, we find that in the ADP-bound actin trimer, the folded DB loop is stable and in a free-energy minimum. It is also demonstrated that the nucleotide binding cleft favors a closed conformation for the bound nucleotide in the ATP and ADP-Pi states, whereas the ADP state favors an open confirmation, both in the monomer and trimer. These results suggest a mechanism of allosteric interactions between the nucleotide binding cleft and the DB loop. This behavior is confirmed by an additional simulation that shows the folding free-energy as a function of the nucleotide cleft width, which demonstrates that the barrier for folding changes significantly depending on the value of the cleft width.

Fig. 1.

Systems and biological events investigated using MD and metadynamics. (A) The actin trimer with the binding pocket and DB loop labeled as α and β, respectively. The monomer shown in blue is the monomer to which metadynamics was applied. (B) Shown is the proposed conformational change of the DB loop. (C) Shown is the nucleotide binding pocket. The arrows in C illustrate the collective variables used to study the binding pocket.

Fig. 2.

Free-energy surfaces for the opening and closing of the nucleotide binding cleft in monomeric actin. The isolines are drawn using a 1 kcal/mol spacing, and the energy scale is in kcal/mol. Based on block-averaging analysis, the uncertainty is ≈1 kcal/mol. The collective variables used in the opening and closing simulations are described in Results.

Fig. 3.

Free-energy profiles for folding the DB loop region in monomeric actin as a function of the bound nucleotide. The profiles are a projection of both path collective variables onto the s collective variable. The transition states in the nucleation-condensation model discussed in Results are labeled TS1 and TS2 and shown (Upper) in ribbon representation along with representative unfolded and folded structures. (Lower) The shaded region marks the approximate location of the fine-grained/coarse-grained border in the path collective variables. The approximate error is 0.6 kcal/mol.

Fig. 4.

Free-energy profiles for folding the DB loop region in the actin trimer as a function of the bound nucleotide for the Holmes (dashed line) and Oda (solid line) filament models. The profiles are a projection of both path collective variables onto the s collective variable. (Upper) Representative unfolded, transition state, and folded structures are given. (Lower) The shaded region marks the approximate location of the fine-grained/coarse-grained border. The approximate error is 0.6 kcal/mol.

Fig. 5.

Free-energy profile for combined metadynamics simulation by using both the cleft width collective variable and the path collective variable (s). The isolines are drawn using a 1-kcal/mol spacing, and the energy scale is in kcal/mol. The collective variables are described in Results.