Structural states and dynamics of the D-loop in actin

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Actin conformational changes driven by ATP hydrolysis regulate actin networks.
• The DNase I binding loop (D-loop) of actin is implicated in nucleotide-dependent conformational changes.

Purpose of the Study:

• To investigate the structural and conformational states of the actin D-loop in solution.
• To determine the secondary structure and nucleotide-independent conformational dynamics of the D-loop in monomeric and filamentous actin.

Main Methods:

• Cysteine scanning mutagenesis and site-directed labeling of actin.
• Acrylodan fluorescence spectroscopy to probe accessibility and environment.
• Electron paramagnetic resonance (EPR) spectroscopy to assess spin-label mobility and conformational states.
• Analysis of a novel actin crystal structure revealing D-loop conformation.

Main Results:

• D-loop cysteine mutants showed no evidence of alpha-helical structure in monomeric or filamentous actin, regardless of nucleotide state.
• Transition from monomeric to filamentous actin resulted in blue-shifted acrylodan emission and increased immobilization of spin labels, particularly at residues 43-47.
• Complex EPR line shapes indicated multiple conformational states for spin-labeled D-loop mutants.
• A new crystal structure revealed the D-loop in a hairpin conformation.

Conclusions:

• The actin D-loop does not adopt an alpha-helical structure.
• In filamentous actin, the D-loop exists in dynamic equilibrium among several conformations, irrespective of the bound nucleotide.
• These findings suggest a flexible and conformationally diverse role for the D-loop in actin regulation.