Gain-of-Function Mutations Conferring Actin-severing Activity to Human Macrophage Cap G

Nonmuscle cell motility requires marked changes in the consistency and shape of the peripheral cytoplasm. These changes are regulated by a gel-sol transformation of the actin filament network, and actin filament-severing proteins are responsible for network solation. Macrophage Cap G, unlike all other proteins in the gelsolin family, caps but does not sever actin filaments. Two amino acid stretches in Cap G diverge markedly from the severing proteins: 84LNTLLGE and 124AFHKTS. Discrete mutations in Cap G have been generated to determine if these amino acid sequences are critical for actin filament severing. Conversion of 84LNTLLGE to the gelsolin actin-binding helix sequence ( 84LDDYLGG) renders Cap G capable of severing actin filaments (half-maximal severing, 1-2 μM). Adding a second set of mutations, converting 124AFHKTS to 124GFKHV, enhances severing by 10-fold (half-maximal severing, 0.1-0.2 μM). These experiments support a critical role for these two regions in actin filament severing and showcase the power of gain-of-function mutations in clarifying structure-function relationships.