EXPRESSING FUNCTIONAL DOMAINS OF MOUSE CALPONIN - INVOLVEMENT OF THE REGION AROUND ALANINE-145 IN THE ACTOMYOSIN ATPASE INHIBITORY ACTIVITYOF CALPONIN

Previously, we attributed the binding of F-actin to the 38-residue str etch of gizzard calponin encompassing the sequence A145-Y182 and postu lated the hexapeptide motif VKYAEK, representing residues 142-147, as a putative actin-binding site [Mezgueldi, M., Fattoum, A., Derancourt, J. & Kassab, R. (1992) J. Biol. Chern. 267, 15943-15951]. Herein, the nature of the ATPase inhibitory amino acids of calponin and their rel ative position within the actin binding domain was investigated by exp ressing the following fragments of mouse calponin with or without subs titution or deletion of the hexapeptide V142-K147: amino acids 1-228 ( CaP1-228), 45-228 (CaP45-228), 131-228 (CaP131-228), and CaP1-228 with substitution of A145 with S (CaP1-228A145S) or deletion of V142-K147 (CaP1-228del142-147). All the recombinant fragments displayed most of the biochemical properties of the smooth muscle purified calponin incl uding (a) expected electrophoretic mobility, (b) heat stability, (c) b inding to actin, tropomyosin and calmodulin, and (d) zero-length cross -linking to actin switched by calmodulin in a calcium-dependent fashio n. However, while the wild-type recombinant fragments inhibit the acto -S-1 ATPase activity to the same extent as do the parent calponin, mod ulation of the hexapeptide by either substitution or deletion strongly affect the inhibitory activity with only slightly decreasing actin bi nding capacity. The data indicate that the stretch VKYAEK is crucial f or ATPase inhibition by calponin but represents only part of the actin -binding domain. These results are discussed in terms of multiple cont act sites between actin and calponin.