Actobindin binds with high affinity to a covalently cross-linked actin dimer

Area of Science:

• Biochemistry
• Cell Biology
• Protein-Actin Interactions

Background:

• Actobindin is a 9.8-kDa protein from Acanthamoeba castellanii.
• It possesses two actin-binding sites, capable of binding two actin monomers simultaneously.
• Actobindin's potent inhibition of actin polymerization exceeds predictions based on its affinity for monomers.

Purpose of the Study:

• To investigate the mechanism behind actobindin's strong inhibition of actin polymerization.
• To test the hypothesis that actobindin interferes with actin polymerization nucleation by binding to actin oligomers.
• To explore the thermodynamic favorability of actobindin binding to oligomers versus monomers.

Main Methods:

• Utilized covalently cross-linked actin dimers to study actobindin binding.
• Measured the binding affinity (apparent KD) of actobindin to cross-linked actin dimers.
• Investigated the specificity of actobindin binding using isomeric cross-linked actin dimers.

Main Results:

• Actobindin exhibits high affinity (apparent KD = 11 nM) for a specific conformation of cross-linked actin dimer.
• This binding affinity supports the hypothesis that actobindin binds to native actin oligomers.
• Actobindin's interaction is specific, with weak binding to an isomeric dimer, which is preferentially formed during polymerization in its presence.

Conclusions:

• Actobindin likely inhibits actin polymerization by binding to native actin oligomers, thereby preventing nucleation.
• The binding mechanism may involve blocking cross-linking sites or inducing a conformational change in the actin dimer.
• The study resolves the paradox of actobindin's potent inhibition through oligomeric interactions.