Crystal Structure of Monomeric Actin in the ATP State

Abstract

A nucleotide-dependent conformational change regulates actin filament dynamics. Yet, the structural basis of this mechanism remains controversial. The x-ray crystal structure of tetramethylrhodamine-5-maleimide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-Å resolution. A comparison of this structure to that of tetramethylrhodamine-5-maleimide-actin with bound ADP, determined previously under similar conditions, reveals how the release of the nucleotide ␥-phosphate sets in motion a sequence of events leading to a conformational change in subdo- The actin filament (F-actin) is asymmetric, undergoing net incorporation of ATP-actin monomers to the barbed end and dissociation of ADP-actin monomers from the pointed end (1, 2). This dynamic process, known as actin filament treadmilling, is an essential part of many forms of cell motility. A driving force behind actin treadmilling is the hydrolysis of ATP by actin. However, in vivo treadmilling is further regulated by a number of factors including a battery of actin-binding proteins. Actin-depolymerizing factor/cofilin, for instance, binds preferentially to the ADP-actin monomers that accumulate toward the pointed end of the filament, accelerating their dissociation. Other proteins such as profilin and thymosin-␤4 bind ATPactin with higher affinity than ADP-actin, maintaining a pool of ATP-actin monomers ready for incorporation into the barbed end of the filament. The fact that these proteins can "distinguish" between ATP-and ADP-actin suggests that these two states are structurally different. Consistent with this view, biochemical (3-5), spectroscopic (6 -8), and electron microscopic (9) evidence has suggested that a conformational change in actin subdomain 2 accompanies the hydrolysis of ATP and the release of inorganic phosphate.

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

“…This region is in the area in subdomain 2 involved in ATP binding, and it also contains the so-called sensory loop, residues 70 -78 (38). His 73 in the sensory loop is methylated in muscle actin but not in yeast or either of the two hybrids.…”

Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments

“…Crystallographic studies of monomeric actin indicate that release of the ␥-phosphate of ATP initiates a series of conformational changes that results in conversion of the disordered DNase I-binding loop of G-ATP-actin to an ␣-helix in G-ADP-actin (23,24). Molecular dynamic simulations of actin oligomers and filaments (25) are consistent with a model in which the helical loop of the ADP-actin subunit weakens actinactin interactions in trimeric nuclei and in F-actin, leading to destabilization of both.…”

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

“…The crystal structure of both G-ADP and G-ATP-actin, always as a complex with another protein or with a covalent modification to prevent polymerization, has been determined more than 30 times (27), and the D-loop has usually been found to be disordered in both G-ATP-and G-ADP-actin. Recently, in crystals of G-actin rendered non-polymerizable by reaction of tetramethylrhodamine 5-maleimide with Cys-374, the D-loop was found to be folded into an ␣-helix in the ADP state (28), although this has been questioned (29), but to be disordered in the ATP state (30). There are five other reports of crystal structures of actin complexes with the D-loop in an ordered conformation, not necessarily ␣-helical (see references within Ref.…”