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 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.

Keywords: autoproteolysis; calcium-binding protein; calpain-3; crystal structure; drug design; propeptide; protease inhibitor; protein complex; small-angle X-ray scattering (SAXS).

Figure 1.

Schematic structures of conventional calpains (calpain-1 and -2) and calpain-3. A, conventional calpain-1 and -2 form heterodimers through the penta-EF-hand domains of the large PEF(L) and small PEF(S) subunits. B, calpain-3 forms a homodimer through PEF(L) domain connections and has unique sequences of NS, IS1, and IS2 shown in gray. The protease core domains (PC1 and PC2) are colored orange and yellow, respectively. C, H, and N, the catalytic triad residues, Cys, His, and Asn. The red section preceding PC1 is the anchor helix that contacts the small subunit. Other domains are the calpain β-sandwich domain (CBSW) in green; the PEF(L) domain in light blue; the glycine-rich domain in pink; and PEF(S) in purple. Numbers, residues at the domain boundaries. Arrows, autolysis sites (1b and 2b) in IS1.

Figure 2.

Prediction of disorder in the calpain-3 protease core sequence. The calpain-3 protease core sequence, including NS, was analyzed for the probability of disorder using a web-based program (http://bioinf.cs.ucl.ac.uk/psipred)³ (27, 32). The likelihood of a region being disordered is plotted as a confidence score against the amino acid sequence. The blue tracing represents sequence in a disordered state, and the orange tracing corresponds to disordered residues likely to be involved in protein-protein interactions.

Figure 3.

Crystal structures of calpain-3 protease core C129S (A) and Cys¹²⁹ in complex with E-64 (B). Ribbon tracings colored orange and yellow represent domains PC1 and PC2 of the calpain-3 protease core, respectively. Residual IS1 in A and B is colored blue. Green stick, E-64 in B. Catalytic residues and the visible N and C termini of IS1 and the protease core are labeled with their residue numbers. Magenta spheres, Ca²⁺.

Figure 4.

Time course of autoproteolysis of calpain-3 protease cores analyzed by SDS-PAGE. Lanes kDa, M, and C, molecular masses, molecular mass markers, and a control protease core without proteolysis (day 0), respectively. All protein samples were incubated in 0.1 m MES (pH 6.5) and 0.1 m CaCl₂. A, analysis of C129S core crystals. B, C129S core in solution. C, C129A core in solution. D–I, C129S core in solution incubated with the protease inhibitors or chemicals indicated below the gel images. Band intensity as measured by densitometry is shown below the gel images of B–I. Blue, orange, gray, and yellow lines, 43, 26.6, 17, and 13 kDa bands, respectively. The time course of the incubations was measured in days (from 1 to 28) indicated above the gel images.

Figure 5.

SAXS analyses of Ca²⁺-free and Ca²⁺-bound calpain-3 C129A protease core. Top, Guinier plot scattering patterns of the C129A calpain-3 core. Ca²⁺-free and Ca²⁺-bound samples are in green and red, respectively. Bottom, pair distribution functions P(r) of Ca²⁺-free (green) and Ca²⁺-bound (red) samples. Both samples give similar maximum dimensions (D_max) for the particles (84.7 Å for Ca²⁺-free and 83.7 Å for Ca²⁺-bound), but the P(r)-distribution profiles are different.

Figure 6.

Agreement between structural models and SAXS experimental scattering patterns for the calpain-3 core. A and B, theoretical scattering patterns calculated by CRYSOL from structural models based on the ensemble optimization method for the Ca²⁺-free and Ca²⁺-bound calpain-3 cores, respectively. C and D, show solution structural models fit to envelopes, which correspond to Ca²⁺-free and Ca²⁺-bound sample, respectively. Green ribbon, PC1 and PC2 domains of the calpain-3 core. Red spheres represent IS1 residues. N and C termini of the core are indicated by N and C, respectively.

Figure 7.

Calpain-3 core structures derived from SAXS analysis. The C129A calpain-3 protease core solution structure is shown in a surface representation with PC1 colored orange, PC2 colored yellow, and IS1 in red. A and B, Ca²⁺-free structure shown with a 40° difference in orientation. C and D, the Ca²⁺-bound structure in the same orientations as in A and B, respectively.

Figure 8.

Contacts between IS1 and residues of calpain-3 C129S protease core active-site cleft. Ribbon and stick in orange represent PC1, and yellow represents PC2. The ribbon and stick in green represent IS1. The black dashed lines indicate hydrophilic interactions between residues. A, active-site cleft from S5′ to S3 subsites; B, active-site cleft from S1′ to S3 subsites.

Figure 9.

Interactions of inhibitors in the active site of the calpain-3 protease core. A, E-64 bound to the active-site cleft of calpain-3 protease core. B, leupeptin bound to the active-site cleft of calpain-3 protease core. Domains PC1 and PC2 of the calpain-3 core are colored orange and yellow, respectively. The cyan stick in A is E-64, and the gray stick in B is leupeptin. Black dashes indicate the electrostatic interactions between E-64/leupeptin and residues in active site of the calpain-3 core. Magenta spheres represent Ca²⁺.

Figure 10.

Covalent attachment of inhibitors to the catalytic Cys¹²⁹ of the calpain-3 core. E-64 (A) and leupeptin (B) are shown in green; Cys¹²⁹ is in gray. Bonds between inhibitors and Cys¹²⁹ are in yellow. Oxygen atoms are red, and nitrogen atoms are blue. The light blue mesh shows the 2F_o − F_c electron density map contoured at the 1σ level.