A Nucleotide State-sensing Region on Actin

Abstract

The nucleotide state of actin (ATP, ADP-P i , or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165-172) changes from an unstructured loop to a ␤-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277-1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P i state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G-and F-actin with actin-binding proteins.Actin, one of the most abundant and conserved eukaryotic proteins, is involved in a variety of cellular functions, from cell division and migration to intracellular transport and endo-and exocytosis. Under physiological conditions, monomeric actin (G-actin) 3 and filamentous actin (F-actin) are in a dynamic equilibrium, strongly favoring the latter. Very low basal ATPase activity of G-actin is amplified strongly upon its polymerization, and the release of inorganic P i in general follows the polymerization with some delay (2, 3). Consequently, under equilibrium conditions, a typical actin filament contains ATP protomers at the growing end followed by an ADP-P i -enriched region and then by an extended ADP region in the rest of the filament. The nature of the bound nucleotide influences the properties of G-and F-actin and controls their interactions with a number of actin-binding proteins (ABP), which play key regulatory and structural roles in the dynamics and maintenance of the actin cytoskeleton. Nucleotide-sensitive ABPs regulate nucleotide exchange rates and the polymerization of G-actin, the nucleation of new filaments, and disassembly of mature filaments. Thus, preferential binding of the Arp2/3 nucleating complex to ATP and ADP-P i F-actin, cofilin to ADP-F-actin, and profilin to ATP-G-actin leads to the acceleration of directe...

Mutant Profilin Suppresses Mutant Actin-dependent Mitochondrial Phenotype in Saccharomyces cerevisiae

“…However, hydrogen-deuterium exchange results demonstrate that the A167E mutation causes conformational changes extending from the W-loop to the top of subdomains 2 and 4 at the pointed end of the protein. The W-loop is conformationally and functionally connected to the nucleotide-binding site (17,18). Our results showing the effects of the actin mutation on C-terminal F-actin fluorescence are consistent with the idea that W-loop alterations affect the conformation of the filament.…”

“…Our results showing the effects of the actin mutation on C-terminal F-actin fluorescence are consistent with the idea that W-loop alterations affect the conformation of the filament. Consistent with this hypothesis is the observation that two other loop mutations at residues 169 and 170 also lead to filament instability (18).…”

“…The actin thus forms essentially a clamp around these secondary structures of profilin, creating a link from the front to the rear faces of the planar actin structure. Residue 167 is one end of a loop, known as the W-loop (17), which has been shown recently to influence the binding of nucleotide in the interdomain cleft that separates the two halves of the actin protein (18). It also lies very near a patch of hydrophobic residues called the "hot spot" (19) that constitutes part of a binding site for a number of actin regulatory proteins besides profilin, such as cofilin (20), twinfilin (21), CapZ (22), formin (23), and proteins containing a WH2 domain(s) (24).…”

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

“…6A). ⑀-ATP that was bound to WT actin was released at a rate of 0.012 Ϯ 0.0029 s Ϫ1 , which is consistent with previous measurements using skeletal actin (28,29). In contrast, ⑀-ATP bound to monomeric D11Q actin was released at an ϳ40-fold faster rate of 0.42 Ϯ 0.098 s Ϫ1 .…”

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