Crystal Structure of Monomeric Actin in the ATP State: STRUCTURAL BASIS OF NUCLEOTIDE-DEPENDENT ACTIN DYNAMICS

Philip Graceffa,Roberto Dominguez

doi:10.1074/jbc.m303689200

Philip Graceffa, Roberto Dominguez

Open Access

https://doi.org/10.1074/jbc.m303689200

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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-A 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 gamma-phosphate sets in motion a sequence of events leading to a conformational change in subdomain 2. The side chain of Ser-14 in the catalytic site rotates upon Pi release, triggering the rearrangement of the loop containing the methylated His-73, referred to as the sensor loop. This in turn causes a transition in the DNase I-binding loop in subdomain 2 from a disordered loop in ATP-actin to an ordered alpha-helix in ADP-actin. Despite this conformational change, the nucleotide cleft remains closed in ADP-actin, similar to ATP-actin. An analysis of the existing structures of members of the actin superfamily suggests that the cleft is open in the nucleotide-free state.

Highlights

Visualization of the structural details of such a conformational change has come from a comparison of crystal structures of the actin monomer (G-actin) in the ATP and ADP states
A nucleotide-dependent conformational change regu- guish” between ATP- and ADP-actin suggests that these two lates actin filament dynamics
The x-ray biochemical [3,4,5], spectroscopic (6 – 8), and electron microcrystal structure of tetramethylrhodamine-5-maleim- scopic [9] evidence has suggested that a conformational change ide-actin with bound AMPPNP, a non-hydrolyzable ATP analog, was determined to 1.85-Å resolution

Summary

Crystal Structure of Monomeric Actin in the ATP State

Other proteins such as profilin and thymosin-␤4 bind ATP- responsible for the conformational change observed in actin actin with higher affinity than ADP-actin, maintaining a pool subdomain 2 [17, 19] According to this hypothesis, long range of ATP-actin monomers ready for incorporation into the barbed allosteric effects due to the binding of the TMR probe to the C end of the filament. It can be asserted that the differences observed between the structures of monomeric actin in the ATP and ADP states [16] are due to the release of the nucleotide ␥-phosphate and not because of crystal packing contacts or the binding of the TMR probe

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

TABLE I Data collection and refinement statistics

CONCLUSIONS