Nucleotide regulation of the structure and dynamics of G-actin

Abstract

Actin, a highly conserved cytoskeletal protein found in all eukaryotic cells, facilitates cell motility and membrane remodeling via a directional polymerization cycle referred to as treadmilling. The nucleotide bound at the core of each actin subunit regulates this process. Although the biochemical kinetics of treadmilling has been well characterized, the atomistic details of how the nucleotide affects polymerization remain to be definitively determined. There is increasing evidence that the nucleotide regulation (and other characteristics) of actin cannot be fully described from the minimum energy structure, but rather depends on a dynamic equilibrium between conformations. In this work we explore the conformational mobility of the actin monomer (G-actin) in a coarse-grained subspace using umbrella sampling to bias all-atom molecular-dynamics simulations along the variables of interest. The results reveal that ADP-bound actin subunits are more conformationally mobile than ATP-bound subunits. We used a multiscale analysis method involving coarse-grained and atomistic representations of these simulations to characterize how the nucleotide affects the low-energy states of these systems. The interface between subdomains SD2-SD4, which is important for polymerization, is stabilized in an actin filament-like (F-actin) conformation in ATP-bound G-actin. Additionally, the nucleotide modulates the conformation of the SD1-SD3 interface, a region involved in the binding of several actin-binding proteins.

Figure 1

The 12-site CG model contains four main sites, representing the cores of the subdomains of actin, and eight minor sites, representing regions that are highly conformationally mobile, functionally important, or solvent exposed. Mapping from the all-atom (a) to CG (b) representation is shown, along with the two collective variables that are biased during US: the dihedral twist and the cleft width. To see this figure in color, go online.

Figure 2

The 2D free-energy surface of the actin monomer as a function of both the dihedral twist and the nucleotide cleft width reveals that both collective variables are affected by the bound nucleotide, and that ADP-bound actin is more conformationally mobile in both the Oda and G-actin conformations. The line in the Oda-ADP panel indicates the transition path between G-actin and Oda structures, as identified using the string method with a double-well CG potential. To see this figure in color, go online.

Figure 3

The interface between SD2 and SD4 is affected by both the nucleotide and the starting configuration. (a) Backbone structure of the average structure from the lowest-energy US window for each system after aligning the internal reference frame of SD4. (b–e) Contacts between SD2 and SD4 in G-ATP, Oda-ATP, G-ADP, and Oda-ADP, respectively. To see this figure in color, go online.

Figure 4

The relative position of SD4 changes significantly in the Oda ADP-bound actin system compared with the other systems simulated. (a) Backbone structure of the average position of the lowest-energy US window for each system after aligning the internal reference frame for SD3, with SD2 removed for clarity. (b and c) Contacts between SD3 and SD4 for Oda ATP and Oda ADP, respectively. To see this figure in color, go online.

Figure 5

The hydrophobic region between SD1 and SD3 adapts to a range of different twists. (a) The backbone structures of the average position of the lowest-energy sampling window for each system after aligning the internal reference frame for SD1, with SD4 removed for clarity. (b–e) Contacts between SD1 and SD3 in G-ATP, Oda-ATP, G-ADP, and Oda-ADP, respectively. To see this figure in color, go online.