Functional Characterization of Disease-Related Human β-Actin Mutants

Mutations in sarcomeric actins have been linked to various skeletal and cardiac myopathies. In contrast, only a small number of mutations in the cytoplasmic isoforms β-actin and γ-actin have been identified and linked to disease processes. Here, we describe the characterization of disease-related human β-actin mutants R183W and E364K. Mutation R183W is associated with developmental malformations, deafness and dystonia. The residue is located in subdomain 4 in the nucleotide binding cleft and participates in forming a gate-like structure for the nucleotide. Mutation E364K was found in a patient with neutrophil dysfunction. E364 is a surface residue in subdomain 1. We produced both mutants in Sf9 cells and characterized their properties in vitro. The midpoints of the thermal transition Tm, which correspond to the temperatures at which half of all protein molecules are in the native state and the remaining half is in the denatured state, are unaffected by the mutations suggesting proper folding and normal protein stability. Both mutants display increased affinity for DNase-I indicating conformational changes in subdomain 2 that appear to alter the binding interface between monomers. Activated neutrophils are highly motile phagocytic cells and they are thus highly sensitive to changes that affect actin polymerization and F-actin stability. We observed reduced rates of nucleotide exchange and elongation velocities during polymerization for R183W and E364K β-actin. Mutation R183W accelerates ATP hydrolysis by F-actin. R183W β-actin shows impaired ability to activate non-muscle myosin 2A ATPase.