Contribution of Distinct Structural Elements to Activation of Calpain by Ca2+ Ions

The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a Kd of 3400 μm. Phospholipids lower this value to 220 μm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 μm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this “extended transducer” mechanism is presented. The effect of Ca2+ in calpain activation is mediated via several binding sites in the enzyme molecule. To test the contribution of structural elements suspected to be part of this Ca2+ relay system, we made a site-directed mutagenesis study on calpains, measuring consequential changes in Ca2+ binding and Ca2+ sensitivity of enzyme activity. Evidence is provided for earlier suggestions that an acidic loop in domain III and the transducer region connecting domains III and IV are part of the Ca2+ relay system. Wild-type Drosophila Calpain B domain III binds two to three Ca2+ ions with a Kd of 3400 μm. Phospholipids lower this value to 220 μm. Ca2+ binding decreases in parallel with the number of mutated loop residues. Deletion of the entire loop abolishes binding of the ion. The Ca2+ dependence of enzyme activity of various acidic-loop mutants of Calpain B and rat m-calpain suggests the importance of the loop in regulating activity. Most conspicuously, the replacement of two adjacent acidic residues in the N-terminal half of the loop evokes a dramatic decrease in the Ca2+ need of both enzymes, lowering half-maximal Ca2+ concentration from 8.6 to 1.3 mm for Calpain B and from 250 to 7 μm for m-calpain. Transducer-region mutations in m-calpain also facilitate Ca2+ activation with the most profound effect seen upon shortening the region by deletion mutagenesis. All of these data along with structural considerations suggest that the acidic loop and the transducer region form an interconnected, extended structural unit that has the capacity to integrate and transduce Ca2+-evoked conformational changes over a long distance. A schematic model of this “extended transducer” mechanism is presented. Calpains are a family of calcium-dependent cytoplasmic cysteine proteinases, regulatory enzymes transducing intracellular Ca2+ signals into limited proteolysis of their substrate proteins. Members of this family are thought to have diverse and basic functions in the regulation of various cellular processes such as cytoskeletal remodeling, cell cycle, apoptosis, and motility (1Sorimachi H. Ishiura S. Suzuki K. Biochem. J. 1997; 328: 721-732Crossref PubMed Scopus (622) Google Scholar, 2Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (341) Google Scholar, 3Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (142) Google Scholar, 4Ohno S. Emori Y. Imajoh S. Kawasaki H. Kisaragi M. Suzuki K. Nature. 1984; 312: 566-570Crossref PubMed Scopus (261) Google Scholar, 5Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (108) Google Scholar). Calpains also take part in some pathological processes, such as muscular dystrophy, cataract, diabetes, and Alzheimer's and Parkinson's diseases (6Huang Y. Wang K.K. Trends Mol. Med. 2001; 7: 355-362Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). Distinct isoforms in vertebrates include the ubiquitous μ- and m-calpain-specific and some tissue-specific forms, e.g. muscle-specific p94 (CAPN3), or the stomach-specific nCL-2 and nCL-4. Calpain homologues have also been described in lower organisms such as insects, nematodes, and fungi and, most recently, in plants (7Lid S.E. Gruis D. Jung R. Lorentzen J.A. Ananiev E. Chamberlin M. Niu X. Meeley R. Nichols S. Olsen O.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5460-5465Crossref PubMed Scopus (217) Google Scholar). The mammalian ubiquitous μ- and m-calpains (CAPN1 and CAPN2) are the best-characterized members of the family. They are traditionally held to be heterodimers of a distinct 80-kDa large subunit and a common 30-kDa small subunit. Recently, we have cloned a homologue of the small subunit, which can substitute for the common small subunit in vitro (8Schád E. Farkas A. Jékely G. Tompa P. Friedrich P. Biochem. J. 2002; 362: 383-388Crossref PubMed Google Scholar). μ-Calpain and m-calpain also differ in the concentration of Ca2+ required for their half-maximal activation in vitro, which is ∼50 μm for μ-calpain and 200 μm for m-calpain. Both of these values are far above the Ca2+ concentrations normally available in the cytoplasm. Autolysis, an autoproteolytic process in which the enzyme cuts its N-terminal tail and so becomes active, decreases the Ca2+ requirement (9Baki A. Tompa P. Alexa A. Molnar O. Friedrich P. Biochem. J. 1996; 318: 897-901Crossref PubMed Scopus (101) Google Scholar). On the basis of sequence homology, the large subunit has been divided into four domains (I–IV) and the small subunit has been divided into two domains (V–VI) (4Ohno S. Emori Y. Imajoh S. Kawasaki H. Kisaragi M. Suzuki K. Nature. 1984; 312: 566-570Crossref PubMed Scopus (261) Google Scholar), of which domain II is a papain-like cysteine protease domain, whereas domains IV and VI are calmodulin-like Ca2+-binding domains. The high resolution x-ray structures of rat (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar) and human (11Strobl 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 (320) Google Scholar) m-calpain support this domain assignment and explain the strict Ca2+ dependence of calpain activity. In the absence of Ca2+, the protease domain is separated into two halves by a deep crevice that prevents residues of the catalytic triad from assuming the correct spatial positions for substrate hydrolysis. In this inactive state, interactions with other domains hold the two halves of the catalytic domain apart. Domain IIa is held by the N-terminal α-helix of the large subunit termed domain I (11Strobl 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 (320) Google Scholar) or anchor peptide (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar), and domain IIb is mostly positioned by multiple salt bridges between its clustered Lys226, Lys230, and Lys234 residues and an acidic loop in domain III composed of 8–10 Asp/Glu residues. To explain the activation of the enzyme, it has been suggested that binding of Ca2+ to domains IV and VI induces conformational changes, which relieve the structural constraints on both sides of the catalytic domain, leading to the assembly of the competent active site (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar, 11Strobl 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 (320) Google Scholar). In the absence of the high resolution structure of the Ca2+-bound active form of the enzyme, this mechanism lacks direct structural evidence. Nevertheless, there are several observations that indirectly support this activation model. The isolated catalytic core of the enzyme, “minicalpain,” which is free of the above constraints, is inclined to form the closed, catalytically competent conformation in the presence of Ca2+ (12Moldoveanu T. Hosfield C.M. Lim D. Elce J.S. Jia Z. Davies P.L. Cell. 2002; 108: 649-660Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The two Ca2+ ions bound to two non-EF hand sites in minicalpain play a decisive role in activation, as evidenced by site directed mutagenesis (13Moldoveanu T. Jia Z. Davies P.L. J. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the anchor peptide by upon calpain activation (9Baki A. Tompa P. Alexa A. Molnar O. Friedrich P. Biochem. J. 1996; 318: 897-901Crossref PubMed Scopus (101) Google Scholar). the of domain the from the structural of domain III with in and in a of other J. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). has been suggested that Ca2+ binding by the acidic loop of domain III salt bridges between domains III and IIb to IIa and the active site crevice (11Strobl 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 (320) Google Scholar). In of the salt bridges by mutations of in domain IIb to the Ca2+ requirement of activation C.M. T. Davies P.L. Elce J.S. Jia Z. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). isolated domain III to Ca2+ and in a P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). of these there are three elements of calpain activation, which have been there is direct that the acidic loop of domain III is a Ca2+ binding it has been that Ca2+ binding by this it to the activation of the it has been that the long or transducer (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar, 11Strobl 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 (320) Google Scholar) connecting domains III and IV a role in activation as suggested (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar). To these we the of these two structural elements by mutations the loop region of isolated domain III from Calpain we this region acidic loop is for the Ca2+ binding of this the Ca2+-binding capacity with the of loop we two of domain III with and the other with Ca2+ and the mutations made in the enzymes, Drosophila Calpain B and rat m-calpain The of Ca2+ sensitivity of these enzymes to the importance of Ca2+ binding by the loop region in calpain we the of the transducer by mutagenesis in various The of this is to it can the D. M. Wang K.K. M. E. M. 1997; PubMed Scopus Google Scholar, H. P. Y. J.S. Davies P.L. Elce J.S. M. 1997; PubMed Scopus Google Scholar) P. J.S. P. M. Elce J.S. Biochem. J. 2000; PubMed Scopus Google Scholar) conformational changes of the calmodulin-like domains upon Ca2+ binding the active of the data an “extended transducer” model is for calpain activation in which the acidic loop and the transducer in a and enzymes and from The and E. from from and from from site-directed mutagenesis from an activity of a of All of the other from to Nature. PubMed Scopus Google Scholar). with concentration to Biochem. PubMed Scopus Google Scholar). by of domain III of Drosophila Calpain B as described P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar), whereas rat m-calpain 80-kDa subunit with a and residues of the small subunit of rat m-calpain provided by J. S. Elce of J.S. C. S. Davies P.L. PubMed Scopus Google Scholar). The to Drosophila Calpain B from the by and and The with and and into the sites of the The by the by the mutagenesis to the of isolated domain III of Calpain Calpain and m-calpain and as The for mutagenesis are in and the of mutants can be seen on The of as In the sequence the mutated site by of for site-directed mutagenesis of domain III of Calpain Calpain and m-calpain to the isolated domain III of Calpain whereas B and to Calpain B and sequence positions in enzymes, from the in a and of E. with the in and the 250 of and of isolated domain III of Calpain B to Tompa P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar) with the that to by of by in A mm mm and for in The into A and for Calpain B from both the and the of E. To or Calpain B from the of the by mm for The on and for in mm mm mm mm mm mm and for μm on The for The to a with with the of is The with and mm Calpain B with a of from to 250 mm in Calpain B into and in A mm mm mm and mm Calpain B also from to and Friedrich G. Friedrich P. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In this with mm for The enzyme the as the of or rat m-calpain with mm for The on and for in and for μm on The for The to a of with The with mm and with 250 mm All of the enzyme for as by be with Calpain B domain III with m-calpain domain III of the of the The in of A into the The into of A with mm and concentrations of from the and from the the and for in a so that the of in of The of bound to the from the between the in the and in the in the presence of the concentration of both and the a concentration to domain III as a to and to the concentration of the the of of from a of to Tompa P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). Ca2+ of in A on a of with The for with on a of and with of μm on a for the with and for on x-ray by in a of Calpain activity in a the of to the of the substrate The and in a of in A mm The concentration of the substrate Ca2+ concentration to the value The by the of The free Ca2+ concentration by a Calpain B and, to a with there is a in the to activation G. Friedrich P. J. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) which enzyme activity it a to of The of the as the of the to enzyme activity. activity as the of activity mm free Ca2+ and the enzyme concentration by the Biochem. PubMed Scopus Google Scholar). The of the enzyme to the active enzymes on the enzymes and the on the into A mm the for to and proteins. The on of the of concentrations of substrate for with of m-calpain to the in by the of these we that 250 for μm substrate to the number of catalytic by a concentration of the enzyme for Ca2+ to Domain III of Drosophila Calpain number and of the Ca2+-binding sites of domain III by Domain III can three Ca2+ ions with a Kd of 3400 μm in the absence of and two Ca2+ ions with a Kd of 220 μm in the presence of a in the for The effect of mutations of the acidic loop of domain III be by of the of the mutants Nevertheless, their Ca2+ binding be and with that of domain III by an on the by The and domain III in and on and in A the to the the binding capacity of in a in parallel In of we such a distinct and that domain III of Calpain B has the capacity for binding Ca2+, whereas the deletion which lacks of the acidic of the is to The deletion in which the loop is two acidic to the loop are of the Ca2+-binding in the N-terminal part of the loop have effect on Ca2+ binding mutations in the the binds as Ca2+ as the and binding capacity of The in the of the has binding whereas mutations in the of the binds In the acidic are with the lower the capacity is to from the Ca2+ we can the that the acidic loop of Calpain B domain III and its two residues on the N-terminal are for Ca2+ binding of the binding to and domains III of Calpain B of or domain III on a and over with of Ca2+ bound to the by the Ca2+ binding capacity of by and are in of that of the S.E. of is B binding in a observations are in with we have that isolated domain III of rat m-calpain and Drosophila Calpain B binds Ca2+ in a P. Emori Y. Sorimachi H. Suzuki K. Friedrich P. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). The effect of which the of Ca2+ binding by an of and lower the number of bound ions to is in with data on the which can to three Ca2+ with a effect of J. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). All of these are in and with suggestions that domain III and the acidic loop two to three Ca2+ ions in (11Strobl 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 (320) Google Scholar). of the of Domain III in Calpain B on the Ca2+-binding of domain III we two of mutations in the acidic loop of domain III in Calpain B in the or part of the The enzymes The activity of Calpain B and its mutants Ca2+ concentrations and from these the values of for enzyme activity of the mutations the Ca2+ dependence of enzyme activity. an with an value of 1.3 The can be into two of activity with a of mm and of activity with a of μm. Ca2+ dependence can be in of the multiple Ca2+-binding sites on their Kd and their contribution to the active A of the of the Ca2+ of enzyme is as The two positions are Ca2+ sites and is in the Ca2+ binding of isolated domain In this enzyme a small contribution to the active site which from domains IV and VI and along the transducer to the catalytic the in domains IV and VI have high for Ca2+ the enzyme form be Ca2+ The salt with the of the active the activity in the Ca2+ high Ca2+ the salt is also to enzyme sensitivity and activity of and of Calpain B and m-calpain of values to the with two or three mm Ca2+ in a The to these structural is by enzyme in which in the part of the enzyme a Ca2+ activity to that of the enzyme, of the activity is of the of Domain III in mutations to of Calpain B in the acidic loop both and activity of m-calpain The of m-calpain 250 which is in with values of μm P. A. E. Friedrich P. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and μm C.M. T. Davies P.L. Elce J.S. Jia Z. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The most with which is the m-calpain of the Drosophila has an Ca2+ dependence two of in Ca2+ 7 μm. The Ca2+ sensitivity of the of Calpain In the of to in or to and to in to an enzyme with a value of and Ca2+ requirement is on the basis of the between the or two the conformation and of the other so that can take over the role of The of these mutants are of the activity of the The Ca2+ sensitivity of is to that of the enzyme, to its in the Drosophila enzyme On the other the deletion in which the entire loop is to Ca2+ with μm. enzyme form the the enzyme catalytic and substrate The of substrate a substrate concentration to enzyme for with Ca2+ and substrate in the of the catalytic is the of decrease in the of enzyme, and on The of that of a described calpain form M. H. S. C. M. J. 2002; PubMed Scopus Google Scholar), an protease which of the of support from the of the molecule. the is and is an activity of the of the loop region the acidic loop in domain III is for the activation and also for the structural of The in Domain III an Ca2+ in and importance of the acidic loop in domain III as a Ca2+-binding region is by the site-directed mutagenesis with both Calpain B and m-calpain. The that Ca2+ binding by this region to the activation of that both calpain are to Ca2+ by and the Ca2+ binding of the isolated domain III to the the most profound effect on the Ca2+ sensitivity of the In the residues are with in a the effect of an Ca2+ on the acidic loop in of both the in and its capacity to with domain these that Ca2+ binding by the loop region in is an in data to the importance of the N-terminal half of the loop and this is the Ca2+ site of The are of with salt bridges between domains IIb and data by Hosfield C.M. T. Davies P.L. Elce J.S. Jia Z. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) mutated residues to the loop and adjacent to the loop in m-calpain with be the of their we made multiple Nevertheless, the data support to the the of the acidic loop region of domain III in calpain activation (11Strobl 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 (320) Google Scholar). The III and IV in far of calpain activation by Ca2+ is the D. M. Wang K.K. M. E. M. 1997; PubMed Scopus Google Scholar, H. P. Y. J.S. Davies P.L. Elce J.S. M. 1997; PubMed Scopus Google Scholar) P. J.S. P. M. Elce J.S. Biochem. J. 2000; PubMed Scopus Google Scholar) conformational changes in domains IV and VI over a long the catalytic the of Hosfield (10Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (294) Google Scholar) that these conformational changes are domain IIb via domain III the transducer an extended which domains III and IV A structural as extended as that transduce long conformational via of two is that it as a that is to domain IV and its small conformational In the lower of the transducer to the N-terminal of domain whereas its is by a with and interactions and with domain IV The other mechanism is that the transducer via an it with domain IV in the region upon Ca2+ binding and to a conformation of a mechanism of has been suggested for several such as R. FEBS Lett. 2001; PubMed Scopus Google Scholar) or M. H. S. C. M. J. 2002; PubMed Scopus Google Scholar). this the transducer region small conformational changes into To into these we have site-directed mutagenesis in this region and the changes in the Ca2+ sensitivity of m-calpain in the lower region the and the N-terminal of domain mutants and or deletion of a in the region the and the of domain or with the Ca2+ sensitivity The deletion has been the which to the of the The x-ray structure of the of domain VI in the presence of Ca2+ D. M. Wang K.K. M. E. M. 1997; PubMed Scopus Google Scholar, H. P. Y. J.S. Davies P.L. Elce J.S. M. 1997; PubMed Scopus Google Scholar) that upon Ca2+ the N-terminal and to the transducer in domain IV and the transducer this and on the of domain the of salt bridges between its acidic loop and domain as mutations the region of the transducer with domain IV calpain to Ca2+ and it is also to that the of this and the of the transducer to the activation in Calpain structural that of the is that the transducer the and the in domain and is to the acidic loop via two salt bridges and in we that the two and form a structural and unit and in a and the of domain All of observations with to the transducer region and domain III into this the “extended transducer” mechanism of calpain activation The three elements of this model are the structural of this extended its and Ca2+ binding by the acidic in the of domain IIb leading to calpain are some data in the which also support these The to Ca2+ by C.M. T. Davies P.L. Elce J.S. Jia Z. J. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), which is a of the extended transducer region salt bridges with domain into this model. proteolysis that the sequence to the salt bridges between the acidic loop and domain becomes to upon to Ca2+ T. Hosfield C.M. Jia Z. Elce J.S. Davies P.L. Biophys. 2001; PubMed Scopus Google Scholar). region is also to in which m-calpain C. Biochem. J. PubMed Scopus Google Scholar) and C. Biochem. J. PubMed Scopus Google Scholar, T. J. Full Text PDF PubMed Google Scholar), to the of the extended transducer region upon Ca2+ In on some of as Ca2+ catalytic are the of an of Ca2+-binding Ca2+ The elements of this system, the in domains IV and the non-EF in catalytic domain the acidic loop in domain and the extended to activation in and to the of J. S. Elce for the for rat m-calpain.

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