ATP and ADP actin states

Abstract

This minireview is dedicated to the memory of Henryk Eisenberg and honors his major contributions to many areas of biophysics and to the analysis of macromolecular states and interactions in particular. This work reviews the ATP and ADP states of a ubiquitous protein, actins, and considers the present evidence for and against unique, nucleotide-dependent conformations of this protein. The effects of ATP and ADP on specific structural elements of actins, its loops and clefts, as revealed by mutational, crosslinking, spectroscopic, and EPR methods are discussed. It is concluded that the existing evidence points to dynamic equilibria of these structural elements among various conformational states in both ATP- and ADP-actins, with the nucleotides impacting the equilibria distributions.

FIGURE 1

G-actin structure. Structure of actin monomer is shown in three projections. The flexible segments of actin are colored as follows: the phosphate binding loops P1 (a.a. 11–16) and P2 (a.a. 154–161) are in light and dark blue, respectively; the sensor loop (a.a. 70–78) is in green; the H-plug (a.a. 264–271) is in olive; the D-loop (a.a. 40–51) is in orange; the W-loop (a.a. 165–172) is in red; the V-stretch (a.a. 227–237) is in cyan; the N- and C-terminal regions are colored in yellow and purple, respectively.

FIGURE 2

Schematic representation of actin treadmilling. Under steady state conditions in the presence of ATP, actin protomers containing ADP dissociate predominantly from the minus end of the filament, exchange the nucleotide in the cleft from ADP to ATP, and then ATP-containing protomers associate predominantly at the plus end of the filament. The dissociation from the minus end is potentiated by cofilin; ADP to ATP exchange is promoted in solution by profilin. Both proteins sense nucleotide-dependent conformations of actin. Overall, ATP hydrolysis by filamentous actin fuels the translocation of protomers from one end of the filament to the other.

FIGURE 3

Open and closed cleft conformations of actin. X-ray structures of actin–profilin complexes superimposed in closed (black; PDB:2btf) and open (white; PDB:1hlu) states. The SD3-4 parts of each structure were coaligned to reveal the scissors-like rotation of SD2. Distances between the tips of the phosphate clamping loops P1 an P2 (Cα-atoms of G15 and D157) in both states differ by 2.9 Å (indicated in yellow). Residues Q59 and D211, colored in red and green, respectively, were mutated to cysteines in order to assess conformational transitions in the cleft. Distances between Cα-atoms of these residues are shown in yellow numbers.

FIGURE 4

Crosslinking approach to mapping the nucleotide cleft conformation. Growth of yeast cells with cysteine mutants in the nucleotide binding loop of actin on agar plates (A) and in liquid YPD medium (B). Both single (C59 and C211) and double (C59/C211) cysteine actin mutants supported yeast growth under various conditions, at rates comparable to those of the WT and S374 actin expressing strains. Yeast cells expressing C203 mutant actin, known to have impaired polymerization, were used as a control. C: C59/C211 mutations do not affect nucleotide release rates as compared with WT actin. D: Representative gels of C59/C211 actin crosslinked with MTS reagents in different nucleotide states. The crosslinking was initiated by addition of equimolar amount of MTS reagents to C59C/211 actin in different nucleotide states and was blocked after one minute by adding N-ethylmaleimide. The intramolecularly crosslinked actin (denoted by X) has higher mobility on SDS-page than uncrosslinked actin (denoted by A). E: Percentage of C59/C211 actin crosslinked by MTS reagents. Bars represent standard errors of three independent experiments.

FIGURE 5

Nucleotide cleft conformation in G- and F-actin. A protomer from a recent high resolution cryo EM reconstructions of F-actin (colored in olive) is superimposed on X-ray structures of G-actin in the open and closed states (PDB:1hlu and 2btf; colored in white and black, respectively). Distances between Cα-atoms of G15-D157 and Q59-D211 are indicated in colors of their parent molecules. In all known X-ray structures of G-actin, the closed state of the phosphate clamp (defined as a distance between G15 and D157) correlates with a closed conformtaion at the mouth of the cleft (typically measured between Q59 and E207; Q59-D211 distance is used here to compare with the results of crosslinking experiments in Figure 4). However in F-actin, open phosphate clamp conformation (7.6 Å) may come with a shorter cleft mouth distance (11.9 Å) than the one in the closed state of G-actin (14.8 Å).

FIGURE 6

X-ray structures of uncomplexed actin in the ATP- and ADP-states. A: The D-loop of TMR-actin adopts α-helical conformation in the ADP- (orange; PDB:1j6z), but not the ATP-state (gray; PDB: 1nwk), suggesting a possible stabilization of the helix by ADP. B: The D-loop of a polymerization incompetent AP actin mutant (A204E/P243K) is disordered both in ATP- and ADP-bound states (PDB:3el2 and 2hf3; colored in gray and orange, respectively).