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. 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. The two ubiquitous calpains, μ- and m-calpain, are cytosolic thiol proteases entirely dependent on Ca2+ for their activity. These two calpains consist of a large or catalytic subunit (80 kDa) (from the genes capn1 and capn2, respectively) and a common small or regulatory subunit (28 kDa) (fromcapn4). The μ- and m-isoforms differ also in the concentration of Ca2+ required for half-maximal activationin vitro. Both enzymes require a Ca2+concentration (∼50 and ∼300 μm for μ- and m-calpain, respectively) that is significantly higher than that available in vivo (<1 μm). Autolysis causes a drop in Ca2+ requirement, but calpain activation in vivo must involve additional factors, such as membrane-binding and activator proteins. Although the physiological roles of these calpains remain unclear, there is much evidence suggesting that they contribute to many cellular processes, including signal transduction, apoptosis, cell cycle regulation, and cytoskeletal reorganization (1Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (619) Google Scholar, 2Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar, 3Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (140) Google Scholar, 4Ohno S. Emori Y. Imajoh S. Kawasaki H. Kisaragi M. Suzuki K. Nature. 1984; 312: 566-570Crossref PubMed Scopus (253) Google Scholar, 5Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (106) Google Scholar). Their physiological importance is exemplified by the recent demonstration that transgenic mice lacking the classical calpain isoforms die during embryonic development (6Arthur J.S. Elce J.S. Hegadorn C. Williams K. Greer P.A. Mol. Cell. Biol. 2000; 20: 4474-4481Crossref PubMed Scopus (294) Google Scholar). Excessive proteolysis by these enzymes in response to altered Ca2+ homeostasis has been observed in several neuropathological states, including Alzheimer's disease (7Wang K.K. Yuen P.W. Adv. Pharmacol. 1997; 37: 117-152Crossref PubMed Scopus (94) Google Scholar, 8Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Nature. 2000; 405: 360-364Crossref PubMed Scopus (903) Google Scholar). With regard to other forms of calpain, defects in calpain-3 lead to the development of limb-girdle muscular dystrophy 2A (9Richard I. Broux O. Allamand V. Fougerousse F. Chiannilkulchai N. Bourg N. Brenguier L. Devaud C. Pasturaud P. Roudaut C. Hillaire D. Passos-Bueno M.-R. Zatz M. Tischfield J.A. Fardeau M. Beckmann J.S. Cell. 1995; 81: 27-40Abstract Full Text PDF PubMed Scopus (860) Google Scholar), calpain-10 is linked to type II diabetes (10Horikawa Y. et al.Nature Genet. 2000; 26: 163-175Crossref PubMed Scopus (1252) Google Scholar), and in Caenorhabditis elegans, the protease activity of the calpain homologue TRA-3 is required for sex determination (11Barnes T.M. Hodgkin J. EMBO J. 1996; 15: 4477-4484Crossref PubMed Scopus (90) Google Scholar, 12Sokol S.B. Kuwabara P.E. Genes Dev. 2000; 14: 901-906PubMed Google Scholar). The x-ray structures of rat (13Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar) and human (14Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar) m-calpain in the absence of Ca2+ show that the large subunit consists of a short α-helix at the N terminus; domains I and II, which constitute the protease function; domain III, which resembles a C2-domain; and domain IV, which contains several EF-hands. The small subunit contains domain V, which is glycine- and proline-rich and was either not present (13Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar) or not detectable (14Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar) in the x-ray structure, and domain VI, which contains several EF-hands and is structurally very similar to domain IV. Importantly, the structure revealed that the protease active site is not assembled in the absence of Ca2+, a feature not previously observed in any other cysteine protease. The protease domains (domains I and II) are held apart in the Ca2+-free conformation by interactions within the molecule, which prevent the catalytic triad residues (Cys-105 in domain I and His-262 and Asn-286 in domain II) from assuming the correct conformation to hydrolyze substrates. Activation by Ca2+ must somehow relieve these constraints, permitting domains I and II to move toward each other and form a competent active site. A Ca2+-bound structure of calpain might answer several questions concerning the Ca2+-induced activation mechanism, but this structure has not yet been determined because of significant technical difficulties. Based on the primary sequence alone, it was for a long time assumed that the EF-hand-containing domains IV and VI were the major determinants of the Ca2+ requirement of calpain. Recent studies have shown that Ca2+ binding at EF-hand 3 makes the largest contribution to calpain activation, EF-hand 2 may make some contribution, and the other EF-hands apparently have no direct role in Ca2+ regulation (15Dutt P. Arthur J.S. Grochulski P. Cygler M. Elce J.S. Biochem. J. 2000; 15: 37-43Crossref Google Scholar). Additionally, it has recently been demonstrated that TRA-3 has Ca2+-dependent protease activity although it lacks the EF-hand domain (12Sokol S.B. Kuwabara P.E. Genes Dev. 2000; 14: 901-906PubMed Google Scholar). Thus it is likely that the observed Ca2+ requirement of the whole enzyme is not determined solely by the EF-hands and is greatly affected by interactions elsewhere in the molecule. The x-ray structures revealed two interesting features in domain III that may be very important in this regard (Fig. 1 A). First, domain III was found to be structurally similar to the Ca2+-dependent C2 domains, which are known to promote the phospholipid binding of C2-containing proteins (16Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 17Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). This is consistent with the following two considerations: 1) calpains are thought to translocate to the cell membrane when activated by Ca2+ and 2) the Ca2+ requirement of calpain is greatly reduced in the presence of phospholipids, which appears to be a part of the in vivo activation mechanism of calpain. Second, at the interface between domains II and III, there is a set of electrostatic interactions involving a remarkably acidic loop composed of Glu-392–Asp-400 and Glu-504 in domain III and clustered lysine residues Lys-226, Lys-230, and Lys-234 in domain II (Fig.1 B). All these residues are highly conserved within the known m-calpain sequences. We hypothesize therefore that this salt bridge region exerts a conformational constraint on the movement of domain II and therefore on the assembly of the active site, which will tend to elevate the Ca2+ requirement of the enzyme. By disrupting these salt bridges, the mutations described here were designed to investigate the effects on the Ca2+ requirement of calpain. Our experiments would further address the question of whether these electrostatic interactions directly contribute to maintaining the inactive conformation of calpain. Site-directed mutagenesis was performed on single-stranded DNA derived from pET-24-m-80k-CHis6, using the following antisense primers: 5′-P-cttctggatgatcgagaagagattgggaggagg-3′ (K226S), 5′-P-gagaacctttctcgagagccgactggatgatcttg-3′ (K230S), 5′-P-gagaacctttctcgagagcctcctggatgatcttg-3′ (K230E), 5′-P-aagcagagaacctgactcgagagccttctggatg-3′ (K234S), 5′-P-agcagagaaccttcctcgagagccttctggat-3′ (K234E), and 5′-P-agtcagccttcttgctcgagaagactcgg-3′ (E504S). In all cases, the nucleotide sequence around the mutated site was confirmed by DNA sequencing. The expression and purification of wild-type rat m-calpain and mutant enzymes were performed as previously described (18Elce J.S Hegadorn C. Gauthier S. Vince J.W. Davies P.L. Protein Eng. 1995; 8: 843-848Crossref PubMed Scopus (65) Google Scholar). Coexpression of the constructs pET-24-m-80k-CHis6 (encoding the m-calpain 80-kDa subunit with a C-terminal histidine tag) and pACpET-21k (encoding the C-terminal 184 residues of the regulatory subunit, referred to as domain VI or the 21-kDa subunit) in Escherichia coli strain BL21(DE3) gives rise to an active heterodimer. Enzymes were purified through several columns and finally concentrated by centrifugation using a BioMax 30-kDa molecular mass exclusion device to ∼10 mg/ml in a buffer containing 10 mmdithiothreitol, 50 mm Tris-HCl, pH 7.6, 100 mm NaCl, and 200 μm EDTA. Small aliquots of the enzyme samples (typically 50 μl) were flash frozen in liquid nitrogen and stored at −70 °C. For the measurement of the Ca2+ dependence of calpain activity, a modification of the standard casein assay was used. The duplicate assays contained 4 mg/ml casein, 0.2 m NaCl, 10 mm 2-mercaptoethanol, and 50 mm Tris-HCl, pH 7.6, in a final volume of 100 μl. Net final CaCl2concentrations ranged from 0 μm to 5.0 mm. The reaction was initiated with 4 μl of enzyme sample (see below), and the mixtures were incubated at 25 °C for 30 min before the reaction was terminated by the addition of 70 μl of ice-cold 10% trichloroacetic acid. The resultant mixture was placed on ice for 10 min and centrifuged at 15,000 rpm for 15 min, and the absorbance values of the supernatants were recorded at 280 nm. Immediately prior to the Ca2+ titration, the enzyme aliquots were freshly thawed from −70 °C and diluted in 50 mm Tris-HCl, pH 7.6, to a final enzyme concentration of 0.2 mg/ml (wild type, E504S, K226S, K230S, K234S) or 2 mg/ml (K230E, K234E). The Ca2+concentration required for half-maximal activity with casein as substrate is given as [Ca2+]0.5. This value was calculated by fitting the normalized activity data to the Hill equation y =x n/(k n + x n) where y is the fraction of maximum activity,k = [Ca2+]0.5, nis the Hill constant, and x is [Ca2+]. Crystals of the m-calpain mutant enzymes were grown in conditions very similar to those for wild-type m-calpain (19Hosfield C.M. Ye Q. Arthur J.S. Hegadorn C. Croall D.E. Elce J.S. Jia Z. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1484-1486Crossref PubMed Scopus (10) Google Scholar). X-ray data from the K230E mutant were collected at 1.54 Å at an in-house facility that consists of an MAR Research imaging plate and a Rigaku RU-200 x-ray generator operated at 50 kV and 100 mA. Data for the E504S mutant were collected at beamline A-1 at the Cornell High Energy Synchrotron Source, using monochromatic radiation at 0.925 Å and a Quantum-4 CCD camera from All were frozen in liquid and data were collected at 100 K. x-ray data were using the Z. W. 1997; PubMed Scopus Google Scholar). Both mutants and wild-type m-calpain in for the K230E mutant were a = = = = the for the E504S mutants = = = = the mutant were to the wild-type we were able to the of wild-type m-calpain directly for the of the mutant All was using the P. J.S. J. M. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; PubMed Scopus Google Scholar) with 10% of the from the for the The for the K230E mutant was which to = at The for the E504S mutant was which was reduced to = at The final and the calculated were using the D.E. J. Mol. Google Scholar). wild-type m-calpain and the mutant enzymes were purified on columns to on of on the mutant Enzymes were for their to hydrolyze casein at of The calculated as the basis for the of the Ca2+ requirement of wild-type and mutant enzymes in this In the conditions the for wild-type m-calpain was μm (Fig. which is in with (1Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (619) Google Scholar, 2Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar, 3Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (140) Google Scholar, 4Ohno S. Emori Y. Imajoh S. Kawasaki H. Kisaragi M. Suzuki K. Nature. 1984; 312: 566-570Crossref PubMed Scopus (253) Google Scholar, 5Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (106) Google Scholar). the mutation E504S in domain III the most the to 1 μm, to a in the Ca2+ requirement with wild-type The effects of each of the lysine residues Lys-230, and to serine or effects on the Ca2+sensitivity of the enzyme (Fig. the mutation of Lys-234 reduced the to 1 μm in the the mutation of Lys-234 to glutamic acid resulted in a in the to 3 μm, to a the K230E and mutations the specific activity of the enzyme to with the wild type, the and mutations no significant on specific of mutations on the Ca2+ requirement and specific activity of of specific wild in a whether mutations that might influence the Ca2+ we structure determination of these mutants by x-ray We were in K226S, and E504S mutants in the absence of Ca2+ in conditions very similar to those for wild-type rat m-calpain (19Hosfield C.M. Ye Q. Arthur J.S. Hegadorn C. Croall D.E. Elce J.S. Jia Z. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1484-1486Crossref PubMed Scopus (10) Google Scholar). we have the structures of the K230E and E504S from which for were The structures of these mutants are from wild-type calpain of and Å on for the E504S mutant and the K230E in the of the these two structures were almost to the wild type, we not with the mutants that rise to and assumed that they would have conformational as The mechanism of calpain activation by Ca2+ is a question that has A interesting feature of these enzymes is that vitro for Ca2+ are of higher than Ca2+ found in the This is not by similar of the EF-hand such as which have in within the physiological to calpain EF-hand to forms have been in the Ca2+ requirement (15Dutt P. Arthur J.S. Grochulski P. Cygler M. Elce J.S. Biochem. J. 2000; 15: 37-43Crossref Google Scholar), suggesting that other features contribute to requirement for The x-ray structure of Ca2+-free rat m-calpain revealed several features that to the inactive conformation of the enzyme. is a loop of acidic residues in domain III, with Glu-504, which make several electrostatic with lysine residues Lys-230, on α-helix in domain The structure of human m-calpain an additional between Glu-504 and be that Glu-504 would have the with are not yet available for and calpain but these residues are almost all conserved in both and calpain these domain III interactions are likely to be present in all therefore to that this set of salt bridges might the of domain II to domain I in the process of the active site, to the activation of calpain. The and E504S mutations both significantly reduced the Ca2+ requirement the specific activity of the enzyme. The by the E504S mutation was than that by the This suggests that Glu-504 makes electrostatic and the that the E504S mutation the salt to Lys-234 and The mutation of these salt The structure of E504S is to that of the wild-type enzyme in the inactive demonstrating that the in the Ca2+ requirement observed in E504S is not a of the disruption of the inactive conformation but must involve the of domain movement during interesting was observed with the which reduced the Ca2+ requirement of the enzyme further than the This mutation also reduced the specific activity of the the sensitivity of this region to in the electrostatic This mutant was to a with the results of this that the electrostatic interactions of and with the acidic Glu-392–Asp-400 loop not to be to the activation of calpain by Ca2+ because the and mutations not either the Ca2+ requirement of the enzyme or specific activity. The K230E which is to a between this and the acidic not the Ca2+ requirement of the enzyme. greatly reduce the specific activity of the for that we The structure of K230E in the absence of Ca2+ was to that of wild-type m-calpain, that the mutation not the domain III in the of the enzyme. it has not been to calpain in the presence of Ca2+, the effects of these mutations on the activated of the enzyme be directly the many mutations that have been in m-calpain, the Lys-234 and Glu-504 mutations described here are the described that in a of the Ca2+ The question therefore of a of salt around Glu-504 can have a major at and has very The residue Glu-504 is at of the or that domain IV to domain III, and this is assumed to Ca2+-induced conformation in domain IV to the of the molecule. The evidence therefore suggests that the of the salt bridges at Glu-504 makes the movement of domain II toward domain I it to occur at a Ca2+ can be in Glu-504 is at the of the salt bridge The movement of domain II in the assembly of the active site, to studies by the many thiol protease would be on a point in the Glu-504 given that the Glu-504 and Lys-234 mutations the of domain II with domain III, we would that domain II in move apart from domain III during the activation domain III additional conformational in the absence of a Ca2+-bound the importance of this in the calpain the mutation in μ-calpain, also the Ca2+ requirement not the of domain III to C2 domains, it also be that Glu-504 and the Glu-392–Asp-400 loop are in very similar to the in C2 domains that in Ca2+-dependent phospholipid The Ca2+ requirement of calpain is greatly reduced in the presence of some in and this is assumed to membrane binding in We therefore that Ca2+ and phospholipid binding to this region of calpain these salt some on the movement of domain II and the Ca2+ requirement of the enzyme. of Ca2+ in the acidic loop by the of the acidic residues most or all of the electrostatic interactions with the residues in domain With the of Glu-504, or two such salt by point mutation may not be to relieve the conformational to the movement of domain that a Ca2+-bound structure may not be available in the experiments of the type here will be required to the mechanism of calpain activation by In this we have for the first time calpain mutants that are to significantly of Ca2+ for activation. we have a specific feature from the EF-hand domains that the Ca2+ sensitivity and activation of In of the sequence conservation in this it is that these interdomain interactions are important to the overall Ca2+requirement of the

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.