John S. Elce

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 Ca2+‐induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 Å crystal structure of m‐calpain in the Ca2+‐free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca2+. It also reveals an unusual thiol protease fold, which is associated with Ca2+‐binding domains through heterodimerization and a C2‐like β‐sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca2+‐binding must induce conformational changes that re‐orient the protease domains to form a functional active site. The α‐helical N‐terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca2+‐sensitivity through association with the regulatory subunit. This Ca2+‐dependent activation mechanism is clearly distinct from those of classical proteases.

A Ca2+ Switch Aligns the Active Site of Calpain

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 Alzheimers 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.

Reduced Cell Migration and Disruption of the Actin Cytoskeleton in Calpain-deficient Embryonic Fibroblasts

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The μ- and m-calpains consist of a μ- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained fromCapn4−/− mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4−/− cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4−/− cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, α-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4+/+ andCapn4−/− cells. FAK, α-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in theCapn4−/− cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4−/− embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.

Ubiquitous Calpains Promote Caspase-12 and JNK Activation during Endoplasmic Reticulum Stress-induced Apoptosis

Ubiquitously expressed μ- and m-calpain proteases are implicated in development and apoptosis. They consist of 80-kDa catalytic subunits encoded by the capn1 and capn2 genes, respectively, and a common 28-kDa regulatory subunit encoded by the capn4 gene. The regulatory subunit is required to maintain the stability and activity of μ- and m-calpains. Accordingly, genetic disruption of capn4 in the mouse eliminated both ubiquitous calpain activities. In embryonic fibroblasts derived from these mice, calpain deficiency correlated with resistance to endoplasmic reticulum (ER) stress-induced apoptosis, and this was directly related to a calpain requirement for activation of both caspase-12 and the ASK1-JNK cascade. This study provides compelling genetic evidence for calpains role in caspase-12 activation at the ER, and reveals a novel role for the ubiquitous calpains in ER-stress induced apoptosis and JNK activation.

m-Calpain is required for preimplantation embryonic development in mice

Backgroundμ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice.ResultsTo distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage.ConclusionWe conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Autolysis, Ca2+ Requirement, and Heterodimer Stability in m-Calpain

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 μM. The lower value of [Ca2+]0.5 is therefore a property of the Δ9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.

Molecular cloning and bacterial expression of cDNA for rat calpain II 80 kDa subunit

The complete cDNA of 3.2 kb for rat calpain II large subunit has been constructed from library- and polymerase chain reaction-derived fragments, and sequenced. The cDNA encodes a protein of 700 amino acids having 93% sequence identity with human calpain II, and 61% identity with human calpain I. The gene possesses 21 exons, of which exons 3-21 have been mapped over 33 kb of the rat genome. A new phagemid expression vector was created from pT7-7 by insertion of the f1 origin and mutation of an NdeI to an NcoI site. Rat calpain II cDNA ligated into this vector expressed in Escherichia coli an 80 kDa protein identical in size to highly purified rat calpain II; this protein was specifically recognized on immunoblots by an affinity-purified anti-rat calpain II antibody. This is the second mammalian calpain II large subunit to be fully sequenced, and the first to be artificially expressed.

Mutations in calpain 3 associated with limb girdle muscular dystrophy: analysis by molecular modeling and by mutation in m-calpain.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized by selective atrophy of the proximal limb muscles. Its occurrence is correlated, in a large number of patients, with defects in the human CAPN3 gene, a gene that encodes the skeletal muscle-specific member of the calpain family, calpain 3 (or p94). Because calpain 3 is difficult to study due to its rapid autolysis, we have developed a molecular model of calpain 3 based on the recently reported crystal structures of m-calpain and on the high-sequence homology between p94 and m-calpain (47% sequence identity). On the basis of this model, it was possible to explain many LGMD2A point mutations in terms of calpain 3 inactivation, supporting the idea that loss of calpain 3 activity is responsible for the disease. The majority of the LGMD2A mutations appear to affect domain/domain interaction, which may be critical in the assembly and the activation of the multi-domain calpain 3. In particular, we suggest that the flexibility of protease domain I in calpain 3 may play a critical role in the functionality of calpain 3. In support of the model, some clinically observed calpain 3 mutations were generated and analyzed in recombinant m-calpain. Mutations of residues forming intramolecular domain contacts caused the expected loss of activity, but mutations of some surface residues had no effect on activity, implying that these residues in calpain 3 may interact in vivo with other target molecules. These results contribute to an understanding of structure-function relationships and of pathogenesis in calpain 3.

Calpain Mutants with Increased Ca2+ Sensitivity and Implications for the Role of the C2-like Domain

The ubiquitous calpain isoforms (μ- and m-calpain) are Ca2+-dependent cysteine proteases that require surprisingly high Ca2+concentrations for activation in vitro (∼50 and ∼300 μm, respectively). The molecular basis of such a high requirement for Ca2+ in vitro is not known. In this study, we substantially reduced the concentration of Ca2+ 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 C2-like domain III were disrupted, and the effects of these mutations on activity and Ca2+sensitivity were analyzed. The mutation to serine of Glu-504, a residue that is conserved in both μ- and m-calpain and interacts most notably with Lys-234, reduced the in vitro Ca2+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 Ca2+ requirement of calpain. The structures of the mutants in the absence of Ca2+ were shown by x-ray crystallography to be unchanged from the wild type, demonstrating that the increase in Ca2+ sensitivity was not attributable to conformational change prior to activation. The conservation of sequence between μ-calpain, m-calpain, and calpain 3 in this region suggests that the results can be extended to all of these isoforms. Whereas the primary Ca2+ 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 Ca2+-induced activation of calpain and that they influence the overall Ca2+ requirement of the enzyme.

Roles of individual EF-hands in the activation of m-calpain by calcium.

m-Calpain is a heterodimeric, cytosolic, thiol protease, which is activated by Ca(2+)-binding to EF-hands in the C-terminal domains of both subunits. There are four potential Ca(2+)-binding EF-hands in each subunit, but their relative affinities for Ca(2+) are not known. In the present study mutations were made in both subunits to reduce the Ca(2+)-binding affinity at one or more EF-hands in one or both subunits. X-ray crystallography of some of the mutated small subunits showed that Ca(2+) did not bind to the mutated EF-hands, but that its binding at other sites was not affected. The structures of the mutant small subunits in the presence of Ca(2+) were otherwise identical to that of the Ca(2+)-bound wild-type small subunit. In the whole enzyme the wild-type macroscopic Ca(2+) requirement (K(d)) was approx. 350 microM. The mutations did not affect the maximum specific activity of the enzyme, but caused increases in K(d), which were characteristic of each site. All the EF-hands could be mutated in various combinations without loss of activity, but preservation of at least one wild-type EF-hand 3 sequence was required to maintain K(d) values lower than 1 mM. The results suggest that all the EF-hands can contribute co-operatively to calpain activation, but that EF-hand 3, in both subunits, has the highest intrinsic affinity for Ca(2+) and provides the major driving force for conformational change.