Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain

The ubiquitous calpain isoforms (mu- and m-calpain) are Ca(2+)-dependent cysteine proteases that require surprisingly high Ca(2+) concentrations for activation in vitro ( approximately 50 and approximately 300 microm, respectively). The molecular basis of such a high requirement for Ca(2+) in vitro is not known. In this study, we substantially reduced the concentration of Ca(2+) required for the activation of m-calpain in vitro through the specific disruption of interdomain interactions by structure-guided site-directed mutagenesis. Several interdomain electrostatic interactions involving lysine residues in domain II and acidic residues in the C(2)-like domain III were disrupted, and the effects of these mutations on activity and Ca(2+) sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both mu- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca(2+) requirement for activity by almost 50%. The mutation of Lys-234 to serine or glutamic acid resulted in a similar reduction. These are the first reported cases in which point mutations have been able to reduce the Ca(2+) requirement of calpain. The structures of the mutants in the absence of Ca(2+) were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca(2+) sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between mu-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca(2+) binding is assumed to occur at EF-hands in domains IV and VI, these results show that domain II-domain III salt bridges are important in the process of the Ca(2+)-induced activation of calpain and that they influence the overall Ca(2+) requirement of the enzyme.     

Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation

The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.     

Stomach-specific calpain, nCL-2/calpain 8, is active without calpain regulatory subunit and oligomerizes through C2-like domains

Calpains constitute a family of intracellular Ca(2+)-regulated cysteine proteases that are indispensable in the regulation of a wide variety of cellular functions. The improper activation of calpain causes lethality or various disorders, such as muscular dystrophies and tumor formation. nCL-2/calpain 8 is predominantly expressed in the stomach, where it appears to be involved in membrane trafficking in the gastric surface mucus cells (pit cells). Although the primary structure of nCL-2 is quite similar to that of the ubiquitous m-calpain large subunit, the enzymatic properties of nCL-2 have never been reported. Here, to characterize nCL-2, the recombinant protein was prepared using an Escherichia coli expression system and purified to homogeneity. nCL-2 was stably produced as a soluble and active enzyme without the conventional calpain regulatory subunit (30K). Purified nCL-2 showed Ca(2+)-dependent activity, with half-maximal activity at about 0.3 mM Ca(2+), similar to that of m-calpain, whereas its optimal pH and temperature were comparatively low. Immunoprecipitation analysis revealed that nCL-2 exists in both monomeric and homo-oligomeric forms, but not as a heterodimer with 30K or 30K-2, and that the oligomerization occurs through domains other than the 5EF-hand domain IV, most probably through domain III, suggesting a novel regulatory system for nCL-2.     

Binding of calpain fragments to calpastatin

Their cDNA-derived amino acid sequences predict that the 80-kDa subunits of the micromolar and millimolar Ca(2+)-requiring forms of the Ca(2+)-dependent proteinase (mu- and m-calpain, respectively) each consist of four domains and that the 28-kDa subunit common to both mu- and m-calpain consists of two domains. The calpains were allowed to autolyze to completion, and the autolysis products were separated and were characterized by using gel permeation chromatography, calpastatin affinity chromatography, and sequence analysis. Three major fragments were obtained after autolysis of either calpain. The largest fragment (34 kDa for mu-calpain, 35 kDa for m-calpain) contains all of domain II, the catalytic domain, plus part of domain I of the 80-kDa subunit of mu- or m-calpain. This fragment does not bind to calpastatin, a competitive inhibitor of the calpains, and has no proteolytic activity in either the absence or presence of Ca2+. The second major fragment (21 kDa for mu-calpain and 22 kDa for m-calpain) contains domain IV, the calmodulin-like domain, plus approximately 50 amino acids from domain III of the 80-kDa subunit of mu- or m-calpain. The third major fragment (18 kDa) contains domain VI, the calmodulin-like domain of the 28-kDa subunit. The second and third major fragments bind to a calpastatin affinity column in the presence of Ca2+ and are eluted with EDTA. The second and third fragments are noncovalently bound, so the 80- and 28-kDa subunits of the intact calpain molecules are noncovalently bound at domains IV and VI. After separation in 1 M NaSCN, the 28-kDa subunit binds completely to calpastatin, approximately 30-40% of the 80-kDa subunit of mu-calpain binds to calpastatin, and the 80-kDa subunit of m-calpain does not bind to calpastatin in the presence of 1 mM Ca2+.     

Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites

The active site residues in calpain are mis-aligned in the apo, Ca(2+)-free form. Alignment for catalysis requires binding of Ca2+ to two non-EF-hand sites, one in each of the core domains I and II. Using domain swap constructs between the protease cores of the mu and m isoforms (which have different Ca2+ requirements) and structural and biochemical characterization of site-directed mutants, we have deduced the order of Ca2+ binding and the basis of the cooperativity between the two sites. Ca2+ binds first to the partially preformed site in domain I. Knockout of this site through D106A substitution eliminates binding to this domain as shown by the crystal structure of D106A muI-II. However, at elevated Ca2+ concentrations this mutant still forms the double salt bridge that links the two Ca2+ sites and becomes nearly as active as muI-II. Elimination of the bridge in E333A muI-II has a more drastic effect on enzyme action, especially at low Ca2+ concentrations. Domain II Ca2+ binding appears essential, because Ca(2+)-coordinating side-chain mutants E302R and D333A have severely impaired muI-II activation and activity. The introduction of mutations into the whole heterodimeric enzyme that eliminate the salt bridge or Ca2+ binding to domain II produce similar phenotypes, suggesting that the protease core Ca2+ switch is crucial and cannot be overridden by Ca2+ binding to other domains.     

Crystal structure of a micro-like calpain reveals a partially activated conformation with low Ca2+ requirement

The two Ca2+-dependent cysteine proteases, micro- and m-calpain, are involved in various Ca2+-linked signal pathways but differ markedly in their Ca2+ requirements for activation. We have determined the structure of a micro-like calpain, which has 85% micro-calpain sequence (the first 48 and the last 62 residues of the large subunit are those from m-calpain) and a low Ca2+ requirement. This construct was used because micro-calpain itself is too poorly expressed. The structure of micro-like calpain is very similar in overall fold to that of m-calpain as expected, but differs significantly in two aspects. In comparison with m-calpain, the catalytic triad residues in micro-like calpain, His and Cys, are much closer together in the absence of Ca2+, and significant portions of the Ca2+ binding EF-hand motifs are disordered and more flexible. These structural differences imply that Ca2+-free micro-calpain may represent a partially activated structure, requiring lower Ca2+ concentration to trigger its activation.     

Ca(2+)-induced structural changes in rat m-calpain revealed by partial proteolysis

Partial proteolysis by exogenous proteases in the presence and absence of Ca(2+) was used to map the protease-resistant domains in m-calpain, and to obtain evidence for the conformational changes induced in this thiol protease by Ca(2+). The complication of autoproteolysis was avoided by using the inactive Cys105Ser calpain mutant. Both trypsin and chymotrypsin produced similar cleavage patterns from the large subunit (domains I-IV), while the small subunit (domain VI) was largely unaffected. N-Terminal sequencing of the major products showed that hydrolysis occurred in the N-terminal anchor peptide, which binds domain I to domain VI, at a site close to the C terminus of domain II, and at several sites within domain III. Of particular importance to the overall Ca(2+)-induced conformational changes was the increase in mobility and accessibility of domain III. The same sites were cleaved in the presence and absence of Ca(2+), but with one exception digestion was much more rapid in the presence of Ca(2+). The exception was a site close to residue 255 located within the active site cleft. This site was accessible to cleavage in the absence of Ca(2+), when the active site is not assembled, but was protected in the presence of Ca(2+). This result supports the hypothesis that Ca(2+) induces movement of domains I and II closer together to form the functional active site of calpain.     

Ritonavir does not inhibit calpain in vitro

Ritonavir, an inhibitor of HIV-1 protease, has been reported to also inhibit the Ca2+-dependent cysteine protease, calpain. We have investigated these claims with an in vitro study of the effect of ritonavir on the m-calpain and mu-calpain isoforms. Ritonavir failed to block either autolytic or hydrolytic calpain activity, but remained fully capable of inhibiting the HIV-1 protease. Any calpain-related effects of ritonavir in cells must, therefore, arise by a mechanism other than direct inhibition of calpains.     

Calpains: an elaborate proteolytic system

Calpain is an intracellular Ca(2+)-dependent cysteine protease (EC 3.4.22.17; Clan CA, family C02). Recent expansion of sequence data across the species definitively shows that calpain has been present throughout evolution; calpains are found in almost all eukaryotes and some bacteria, but not in archaebacteria. Fifteen genes within the human genome encode a calpain-like protease domain. Interestingly, some human calpains, particularly those with non-classical domain structures, are very similar to calpain homologs identified in evolutionarily distant organisms. Three-dimensional structural analyses have helped to identify calpain's unique mechanism of activation; the calpain protease domain comprises two core domains that fuse to form a functional protease only when bound to Ca(2+)via well-conserved amino acids. This finding highlights the mechanistic characteristics shared by the numerous calpain homologs, despite the fact that they have divergent domain structures. In other words, calpains function through the same mechanism but are regulated independently. This article reviews the recent progress in calpain research, focusing on those studies that have helped to elucidate its mechanism of action. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.     

Inhibition of calpain by a synthetic oligopeptide corresponding to an exon of the human calpastatin gene

Calpastatin is a widely distributed endogenous inhibitor protein specifically acting on calpain (Ca2+-dependent cysteine endopeptidase). The inhibitor consists of four inhibitory domains (Domains 1-4) with mutually homologous sequences. NH2-terminal Domain L is non-homologous, and all domains have 120-140 residues each. A human calpastatin genomic DNA clone was isolated using a previously obtained human calpastatin cDNA probe. Sequence analysis has revealed that the clone contains Domain 1 and segments of neighboring domains (Domains L and 2). Each of three highly conserved, restricted regions within Domain 1 was located on separate exons, 1A, 1B, and 1C. Exon 2A, corresponding to the first exon of Domain 2, is homologous to Exon 1A and follows Exon 1D of Domain 1. A 27-residue peptide encoded by Exon 1B, including a 12-residue middle conserved sequence, was chemically synthesized and tested for protease inhibitory activities. The synthetic peptide showed strong inhibition against calpain I (low Ca2+-requiring form), and calpain II (high Ca2+-requiring form), but no inhibition against papain or trypsin. These results indicated that Exon 1B forms a self-sufficient functional subdomain of the calpastatin inhibitory domain.     

Dissociation and aggregation of calpain in the presence of calcium

Calpain is a heterodimeric Ca(2+)-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca(2+) on the enzyme include activation, aggregation, and autolysis. They may also include subunit dissociation, which has been the subject of some debate. Using the inactive C105S-80k/21k form of calpain to eliminate autolysis, we have studied its disassociation and aggregation in the presence of Ca(2+) and the inhibition of its aggregation by means of crystallization, light scattering, and sedimentation. Aggregation, as assessed by light scattering, depended on the ionic strength and pH of the buffer, on the Ca(2+) concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca(2+), but this was fully reversible by EDTA. With Ca(2+) in 0.2 m NaCl, no aggregation was visible but ultracentrifugation showed that a mixture of soluble high molecular weight complexes was present. Calpastatin prevented aggregation, leading instead to the formation of a calpastatin-calpain complex. Crystallization in the presence of Ca(2+) gave rise to crystals mixed with an amorphous precipitate. The crystals contained only the small subunit, thereby demonstrating subunit dissociation, and the precipitate was highly enriched in the large subunit. Reversible dissociation in the presence of Ca(2+) was also unequivocally demonstrated by the exchange of slightly different small subunits between mu-calpain and m-calpain. We conclude that subunit dissociation is a dynamic process and is not complete in most buffer conditions unless driven by factors such as crystal formation or autolysis of active enzymes. Exposure of the hydrophobic dimerization surface following subunit dissociation may be the main factor responsible for Ca(2+)-induced aggregation of calpain. It is likely that dissociation serves as an early step in calpain activation by releasing the constraints upon protease domain I.     

Activation of calpain by Ca2+: roles of the large subunit N-terminal and domain III-IV linker peptides

The calpains are a family of cysteine proteases with closely related amino acid sequences, but a wide range of Ca(2+) requirements (K(d)). For m-calpain, K(d) is approximately 325microM, for mu-calpain it is approximately 50microM, and for calpain 3 it is not strictly known but may be approximately 0.1microM. On the basis of previous structure determination of m-calpain we postulated that two regions of the calpain large subunits, the N-terminal peptide (residues 1-20) and a domain III-IV linker peptide (residues 514-530 in m-calpain) were important in defining K(d). The mutations Lys10Thr in the N-terminal peptide, and Glu517Pro in the domain linker peptide, reduced K(d) of m-calpain by 30% and 42%, respectively, revealing that these two regions are functionally important. The increased Ca(2+)-sensitivity of these mutants demonstrate that the Lys10-Asp148 salt link and the short beta-sheet interaction involving Glu517 are factors contributing to the high K(d) of m-calpain. Though these two regions are physically remote from the active site and Ca(2+)-binding site, they play significant roles in regulating the response of calpain to Ca(2+). Differences in these interactions in mu-calpain and in calpain 3 are also consistent with their progressively lower K(d) values.     

A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor

The Ca(2+)-dependent cysteine protease calpain along with its endogenous inhibitor calpastatin is widely distributed. The interactions between calpain and calpastatin have been studied to better understand the nature of calpain inhibition by calpastatin, which can aid the design of small molecule inhibitors to calpain. Here we present the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain VI of calpain. DIC19 is a 19 residue peptide, which corresponds to one of the three interacting domains of calpastatin, which is known to interact with domain VI of calpain. We present two crystal structures of DIC19 bound to domain VI of calpain, determined by molecular replacement methods to 2.5A and 2.2A resolution. In the process of crystallizing the inhibitor complex, a new native crystal form was identified which had the homodimer 2-fold axis along a crystallographic axis as opposed to the previously observed dimer in the asymmetric unit. The crystal structures of the native domain VI and its inhibitor PD150606 (3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid) complex were determined with the help of molecular replacement methods to 2.0A and 2.3A resolution, respectively. In addition, we built a homology model for the complex between domain IV and DIA19 peptide of calpastatin. Finally, we present a model for the calpastatin-inhibited calpain.     

Evolutionary and physical linkage between calpains and penta-EF-hand Ca2+-binding proteins

The name calpain was historically given to a protease that is activated by Ca(2+) and whose primary structure contains a Ca(2+)-binding penta-EF-hand (PEF) as well as a calpain cysteine protease (CysPc) domain and a C2-domain-like (C2L) domain. In the human genome, CysPc domains are found in 15 genes, but only nine of them encode PEF domains. Fungi and budding yeasts have calpain-like sequences that lack the PEF domain, and each protein (designated PalB and Rim13, respectively) is orthologous to human calpain-7, indicating that the calpain-7 orthologs are evolutionarily more conserved than classical calpains possessing PEF domains. An N-terminal region of calpain-7 has a tandem repeat of microtubule-interacting and transport domains that interact with a subset of endosomal sorting complex required for transport (ESCRT) III proteins. In addition to calpains, PEF domains are found in other Ca(2+)-binding proteins including ALG-2 that associates with ALIX (an ESCRT-III accessory protein) and TSG101 (an ESCRT-I subunit). Phylogenetic comparison of dissected domain structures of calpains and experimentally confirmed protein-protein interaction networks imply that there is an evolutionary and physical linkage between mammalian calpains and PEF proteins involving the ESCRT system.     

Sidekicks: synaptic adhesion molecules that promote lamina-specific connectivity in the retina

Ca(2+) signaling by calpains leads to controlled proteolysis during processes ranging from cytoskeleton remodeling in mammals to sex determination in nematodes. Deregulated Ca(2+) levels result in aberrant proteolysis by calpains, which contributes to tissue damage in heart and brain ischemias as well as neurodegeneration in Alzheimer's disease. Here we show that activation of the protease core of mu calpain requires cooperative binding of two Ca(2+) atoms at two non-EF-hand sites revealed in the 2.1 A crystal structure. Conservation of the Ca(2+) binding residues defines an ancestral general mechanism of activation for most calpain isoforms, including some that lack EF-hand domains. The protease region is not affected by the endogenous inhibitor, calpastatin, and may contribute to calpain-mediated pathologies when the core is released by autoproteolysis.     

In vitro Proteolytic Degradation of Bovine Brain Calcineurin by m-Calpain

A major cause of neuronal dysfunction is due to altered Ca2+ regulation. An increase in Ca2+ influx can activate Ca2+-dependent enzymes including calpains, causing the proteolysis of its specific substrates. In the present study, calcineurin (CaN) was found to be proteolysed by a Ca2+-dependent cysteine protease, m-calpain. In the presence of Ca2+, the 60 kDa subunit (CaN A) was degraded to a 46 kDa immunoreactive fragment, whereas in the presence of Ca2+ /calmodulin (CaM) immunoreactive fragments of 48 and 54 kDa were observed. The beta-subunit (CaN B) was not proteolysed in either condition. The proteolysis of CaN A increased its phosphatase activity and rendered it totally CaM-independent after 10 min of proteolysis. The molecular weight of the proteolytic fragments suggested that the m-calpain cleaved CaN A in the CaN B binding domain. A CaM-overlay experiment revealed that the CaM-binding site was present only in the 54 kDa fragment produced by CaN A proteolysis in the presence of Ca2+ /CaM. Thus, the increase in CaN A phosphatase activity observed in many neuronal disorders, may be due to the action of calpain.     

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.     

Domain III of calpain is a ca2+-regulated phospholipid-binding domain

The X-ray structure of m-calpain shows that domain III of the large subunit is structurally related to C2 domains, Ca2+-regulated lipid binding modules in many enzymes. To address whether this structural similarity entails functional analogy, we have characterized recombinant domain III from rat micro- and m-calpain and Drosophila CALPB. In a Ca2+ overlay assay domain III displays a large capacity for Ca2+ binding, commensurable with that of domain IV, the principal Ca2+-binding domain of calpains. The amount of Ca2+ bound to domain III increases 2- to 10-fold upon the addition of liposomes containing 20-40% di- and triphosphoinositides. Conversely, phospholipid-binding in spin-column size-exclusion chromatography is significantly promoted by Ca2+, in a manner similar to known C2 domains. These results suggest that domain III might be the primary lipid binding site of calpain and may play a decisive role in orchestrating Ca2+- and lipid activation of the enzyme.