Crystal Structures of Calpain–E64 and –Leupeptin Inhibitor Complexes Reveal Mobile Loops Gating the Active Site

118

110

Calpains are proteases that catalyze the limited cleavage of target proteins in response to Ca 2؉ signaling. Because of their involvement in pathological conditions such as post-ischemic injury and Alzheimer and Parkinson disease, calpains form a class of pharmacologically significant targets for inhibition. We have determined the sequence preference for the hydrolysis of peptide substrates of the ubiquitous -calpain isoform by a peptide library-based approach using the proteolytic core of -calpain (I-II). The approach, first described by Turk et al. (Turk, B. E., Huang, L. L., Piro, E. T., and Cantley, L. C. (2001) Nat. Biotechnol. 19, 661-667), involved the digestion of an N-terminally acetylated degenerate peptide library in conjunction with Edman sequencing to determine the specificity for residues found at primed positions. The cleavage consensus for these positions was then used to design a second, partially degenerate library, to determine specificity at unprimed positions. We have improved upon the original methodology by using a degenerate peptide dendrimer for determination of specificity at unprimed positions. By using this modified approach, the complete cleavage specificity profile for I-II was determined for all positions flanking the cleaved peptide. A previously known preference of calpains for hydrophobic amino acids at unprimed positions was confirmed. In addition, a novel residue specificity for primed positions was revealed to highlight the importance of these sites for substrate recognition. The optimal primed site motif (MER) was shown to be capable of directing cleavage to a specific peptide bond. Accordingly, we designed a fluorescent resonance energy transferbased substrate with optimal cleavage motifs on the primed and non-primed sides (PLFAER). The -calpain core shows a far greater turnover rate for our substrate than for those based on the cleavage site of ␣-spectrin or the proteolytic sequence consensus compiled from substrate alignments.Calpains (clan CA, family C2 in the MEROPS data base (1)), a family of calcium-activated intracellular proteases, are found in animals, plants, and possibly bacteria (2). They convert intracellular calcium signals (3) into a proteolytic signal by catalyzing the limited cleavage of target proteins (4, 5). Among the known cellular substrates of calpain are numerous cytoskeletal proteins, as well as some receptors and integral membrane proteins like the Na ϩ /Ca 2ϩ exchanger, NCX-3 (6). Calpains must be strictly regulated because they catalyze irreversible processing in the cell. In one scenario, calpains localize to the plasma membrane under activating conditions (7,8). This placement may act to position calpains where they can respond to brief calcium influxes from the opening of calcium channels, resulting in very localized and transient activity. Deactivation of calpain can come about in several ways: binding to the endogenous calpain inhibitor calpastatin (9); autoproteolytic inactivation; or simply the dissipation of local high calcium levels. Unr...

Section: Discussionmentioning

confidence: 56%

Determination of Peptide Substrate Specificity for μ-Calpain by a Peptide Library-based Approach

Cuerrier

Moldoveanu

Davies

2005

118

110

“…Despite this information and the numerous reports on the structural properties of calpain (17)(18)(19)(20)(21)(22), including the presence of different isoforms in various tissues (23)(24)(25)(26)(27), the biological function of this protease and the efficiency of its "in vivo" intracellular regulation remain uncertain.…”

mentioning

confidence: 99%

Regulation of Calpain Activity in Rat Brain with Altered Ca2+ Homeostasis

Averna

Stifanese

Tullio

et al. 2007

The regulatory mechanism of calpain activity is primarily based on the physiological control of intracellular Ca 2ϩ homeostasis, predominantly exerted by the plasma membrane Ca 2ϩ -ATPase and, more specifically, by the expression in all mammalian cells of a natural protein inhibitor named calpastatin (1-7).Calpastatin is a protein endowed with peculiar molecular properties characterized by the presence of four identical inhibitory domains, each one possessing, in its free form, an inhibitory capacity almost equivalent to that expressed by the native calpastatin molecule (2-4, 8 -11). Moreover, calpastatin also behaves as a substrate of calpain, which is degraded to single inhibitory domains as well as to inactive products as a result of more extensive digestion (8,(12)(13)(14)(15)(16). Conservative fragmentation has been attributed to -calpain activity, and degradation to inactive peptides attributed to digestion by m-calpain (8, 16).Despite this information and the numerous reports on the structural properties of calpain (17-22), including the presence of different isoforms in various tissues (23-27), the biological function of this protease and the efficiency of its "in vivo" intracellular regulation remain uncertain.It is often suggested (28 -34) that in brain, calpain is directly implicated in neurodegeneration and neuronal cell death as a consequence of a massive Ca 2ϩ influx occurring under pathological conditions, such as hypoxia and ischemia. This statement has been largely based on the protective effects exerted by synthetic protease inhibitors and by overexpression of calpastatin (35-40). However, in these reports, the fact that a large excess of calpastatin is normally present in brain is not considered, and no explanation has been given on the possible ineffectiveness of the inhibitor under the experimental conditions used (31). It must also be considered that the severe and irreversible cell damage observed under these pathological conditions occurs in the presence of a massive Ca 2ϩ influx, associated with the collapse of the membrane potential. On the basis of these considerations, it seems therefore reasonable to assume that in brain cells, as well as in other cell types, a moderate alteration in Ca 2ϩ homeostasis, even prolonged for a long period of time, could evoke adaptive compensatory defense mechanisms capable of preserving cell integrity.To explore in vivo the existence in the brain of such mechanisms and to characterize their molecular aspects, we have treated normal normotensive Milan strain rats (NMS) 2 with a high salt (sodium) diet (HSD) known to promote elevation in blood pressure in response to a mild increase in intracellular free [Ca 2ϩ ] in vascular smooth muscle, via an alteration in the Na ϩ /Ca 2ϩ exchanger (41)(42)(43). A role of the exchanger in the alteration of calcium homeostasis in brain has been previously suggested (44,45). To amplify the effects of this treatment we have exposed hypertensive rats (HMS) of the same Milan strain (46) to the same diet, because these an...

“…Although E-64 and leupeptin bind in the active site of calpain-3 core after IS1 cleavage, and form a covalent bond to the catalytic Cys129, both exogenous inhibitors have many fewer contacts with residues of the active site than does the N terminus of IS1. The electrostatic interactions made by E-64 and leupeptin in the calpain-3 core are also fewer than those made in the calpain-1 core crystal structures (43). This further suggests that E-64 and leupeptin are not optimal active-site inhibitors of the calpain-3 protease core.…”

Section: Discussionmentioning

confidence: 84%

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

Campbell

Davies

2018

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.