Structures of human calpain-3 protease core with and without bound inhibitor reveal mechanisms of calpain activation

Abstract

Limb-girdle muscular dystrophy type 2a arises from mutations in the Ca 2+ -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 Cys-129 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 Ca 2+ ions to the equivalent sites used in calpain-1 and -2.

Novel CAPN3 variant associated with an autosomal dominant calpainopathy

“…The origin of this modification of activity could be an indirect loss of catalytic activity and possibly a modification of interactions with partners due to conformational changes in the protein structure. Indeed, using the available information regarding the calpain-3 structure, we locate the c.1333G>A [p.(Gly445Arg)] CAPN3 variant in the calpain b-sandwich domain (CBSW) interacting with the penta-EF-hand domain (PEF) supposedly involved in the dimerization of two calpain-3 units [37]. Interestingly, the c.1333G>A [p.(Gly445Arg)] CAPN3 variant is located on a loop of the CBSW domain that has interactions with the PEF domain, and is expected to be involved in the communication of Ca 2+ -induced conformational change throughout these domains.…”

Calcium Mechanisms in Limb-Girdle Muscular Dystrophy with CAPN3 Mutations

“…The three specific regions of CAPN3 (NS, IS1, and IS2) are shown in blue. Schematics have been modified from Ye et al, 2018 [82]. The right panel depicts the estimated tertiary structure of CAPN3.…”

Splicing impact of deep exonic missense variants in CAPN3 explored systematically by minigene functional assay

“…Exon–intron junctions of the CAPN3 gene as well as the functional domains of CALPAIN 3 are visualized. The CALPAIN 3 domain nomenclature is shown according to Ono et al (2016) and Ye et al (2018). (b) Three hundred and eighty‐one clinically relevant CAPN3 variants (pathogenic, likely pathogenic and uncertain/conflicting) from LOVD and ClinVar databases are visualized along the CAPN3 gene with SpliceAI scores on the y ‐axis.…”

“…PP3 was also assigned if two out of three algorithms (HSF, MaxEntScan, SpliceAI) indicated an impact on splicing. PM1 score was assigned for variants affecting highly conserved positions located in the Protease Core subdomains 1 and 2 (PC1 and PC2) of the Calpain‐type cysteine Protease conserved (CysPc) domain (amino acids 48–267, 316–415), in the calpain‐type β‐sandwich domain (CBSW, amino acids 429–586), or in the penta‐EF‐hand domain (PEF, amino acids 647–821; Ono, Ojima, Shinkai‐Ouchi, Hata, & Sorimachi, 2016; Ye, Campbell, & Davies, 2018). The position conservation was visualized by the VarMap web tool (Stephenson et al, 2019).…”