Abstract
Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca2+-activated intracellular cysteine protease calpain-3. This calpain isoform is abundant in skeletal muscle and differs from the main isoforms, calpain-1 and -2, in being a homodimer and having two short insertion sequences. The first of these, IS1, interrupts the protease core and must be cleaved for activation and substrate binding. Here, to learn how calpain-3 can be regulated and inhibited, we determined the structures of the calpain-3 protease core with IS1 present or proteolytically excised. To prevent intramolecular IS1 autoproteolysis, we converted the active-site Cys to Ala. Small-angle X-ray scattering (SAXS) analysis of the C129A mutant suggested that IS1 is disordered and mobile enough to occupy several locations. Surprisingly, this was also true for the apo version of this mutant. We therefore concluded that IS1 might have a binding partner in the sarcomere and is unstructured in its absence. After autoproteolytic IS1 removal from the active Cys129 calpain-3 protease core, we could solve its crystal structures with and without the cysteine protease inhibitors E-64 and leupeptin covalently bound to the active-site cysteine. In each structure, the active state of the protease core was assembled by the cooperative binding of two Ca2+ ions to the equivalent sites used in calpain-1 and -2. These structures of the calpain-3 active site with residual IS1 and with bound E-64 and leupeptin may help guide the design of calpain-3-specific inhibitors.
Highlights
Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca2؉-activated intracellular cysteine protease calpain-3
Most of IS1 was not seen in the crystal structure of human calpain-3 Cys129 mutated to Ser (C129S) protease core
Activating cleavage of the internal IS1 propeptide in calpain-3 is regulated by calcium signaling
Summary
Most of IS1 was not seen in the crystal structure of human calpain-3 C129S protease core. The calpain-3 protease core immediately follows a unique N-terminal sequence of 47 amino acids (NS), which is predicted by PSIPRED [27, 28] to be highly disordered (Fig. 2) Because this region is rapidly autolyzed [26], the first 45 residues of the NS sequence were deleted from the expression construct used here. Thereby provide an opportunity to determine its structure and function within the core, we replaced the catalytically active residue Cys129 with Ser [26] This mutation produced a high yield of soluble protein that was amenable to crystallization. No electron density was seen for the N-terminal residues Ile46–Ile of any molecule or even up to Glu in some molecules (Fig. 3) Whereas this N-terminal end of the core is flexible, the C terminus is more rigid and is only missing the last residue, Asp419, in one of the four molecules.
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