Profilin binding causes a moderate opening of the nucleotide cleft in actin. (A) Superimposition of the structures of profilin–Dictyostelium-actin (blue and cyan) and uncomplexed monomeric actin (28) (blue and magenta). Two orientations are shown, rotated by 90°. The latter structure was obtained by mutagenesis in subdomain 4 and is thought to be free of perturbations resulting from the binding of an ABP or chemical cross-linking. For clarity, profilin is not shown in this figure (see Figs. S5 and S6 for a full view of the profilin–actin structure). Subdomains 3 and 4 of the structures were superimposed (blue) to highlight the relative movement of subdomains 1 and 2 (magenta or cyan). Using the classical view of actin as a reference (left view), the 4.7° rotation (calculated with the program DynDom, http://www.sys.uea.ac.uk/dyndom/) between the two major domains of actin can be visualized as two perpendicular rotations of ≈3.3°. The center of this rotation approximately coincides with the junctions between domains, consisting of residue Lys-336 and the helix between residues Ile-136 and Gly-146. Comparison of the profilin–actin structures with any other structure of actin, except for the wide-open structure of profilin–β-actin (36), results in a similar motion of the two major domains (see also Movies S2 and S3). This movement appears less dramatic than previously anticipated (36, 37), but it is probably sufficient to explain the stimulation of nucleotide exchange by profilin. (B) Quenching of tryptophan fluorescence on profilin binding (the results of two identical experiments, with different preparations of both actins, are shown). Profilin binds pY53-actin and unphosphorylated actin with similar affinities (K_d = 0.090 and 0.057 μM, respectively), but the quenching of tryptophan fluorescence is significantly less for profilin–pY53-actin.

Conformational change in actin subdomain 2 on phosphorylation of Tyr-53. (A and B) Close views of subdomain 2 in the structures of unphosphorylated actin and pY53-actin, showing omit electron density maps (contoured at 1 σ) around Tyr-53 (see Figs. S3 and S5 for a full view of the G1-actin structure). The D-loop was not visualized in the unphosphorylated structure. Hydrogen-bonding contacts (red dashed lines) between the oxygen atoms of the phosphate group on Tyr-53 and residues of the D-loop stabilize the conformation of the D-loop in the structure of pY53-actin. This and other figures of the paper were generated with the program PyMOL (http://pymol.sourceforge.net/). (C) Phosphorylation protects the D-loop from subtilisin cleavage, as shown by the ≈50% decrease in the initial rate of digestion. (D and E) Based on the increase in fluorescence as etheno-ATP replaces actin-bound ATP, phosphorylation reduces the rate of nucleotide exchange from 0.011 s⁻¹ for unphosphorylated actin to 0.006 s⁻¹ for pY53-actin, but profilin accelerates and gelsolin inhibits nucleotide exchange to the same extents for both forms of actin. The increase in fluorescence at equilibrium for pY53-actin is only 50% of the increase for unphosphorylated actin. Data were recorded every 10 s.