Visualizing the elusive open shape of G-actin in solution by SAXS data analysis

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Biochemical data suggests ATP hydrolysis opens the G-actin nucleotide binding cleft.
• Crystal structures of G-actin, however, show a closed conformation irrespective of nucleotide state (ATP/ADP).

Purpose of the Study:

• To reconcile the discrepancy between biochemical data and crystal structures regarding G-actin conformation.
• To investigate the structural impact of ATP hydrolysis on G-actin.

Main Methods:

• Small-angle X-ray scattering (SAXS) analysis of G-actin.
• Kratky analysis to assess particle behavior.
• Shape reconstruction using dummy residues and inertial axes overlay.

Main Results:

• SAXS intensities indicated an increase in G-actin's radius of gyration (R(G)) and maximum linear dimension (D(max)) upon ATP hydrolysis.
• Kratky analysis confirmed G-actin behaves as a globular particle irrespective of bound nucleotide.
• Shape reconstruction revealed a compact ATP/AMP-PNP bound G-actin and an open nucleotide binding site after ATP hydrolysis, similar to beta-actin and hexokinase.

Conclusions:

• ATP hydrolysis induces a conformational change, opening the nucleotide binding cleft in G-actin.
• SAXS and shape reconstruction provide evidence for a nucleotide-dependent structural transition in G-actin.