Dissociation and Aggregation of Calpain in the Presence of Calcium
2001; Elsevier BV; Volume: 276; Issue: 50 Linguagem: Inglês
10.1074/jbc.m105149200
ISSN1083-351X
AutoresGour P. Pal, John S. Elce, Zongchao Jia,
Tópico(s)Connexins and lens biology
ResumoCalpain is a heterodimeric Ca2+-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca2+ 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 Ca2+ 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 Ca2+ concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca2+, but this was fully reversible by EDTA. With Ca2+ 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 Ca2+ 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 Ca2+ was also unequivocally demonstrated by the exchange of slightly different small subunits between μ-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 Ca2+-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. Calpain is a heterodimeric Ca2+-dependent cysteine protease consisting of a large (80 kDa) catalytic subunit and a small (28 kDa) regulatory subunit. The effects of Ca2+ 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 Ca2+ 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 Ca2+ concentration, and on the presence or absence of calpastatin. At low ionic strength, calpain aggregated rapidly in the presence of Ca2+, but this was fully reversible by EDTA. With Ca2+ 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 Ca2+ 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 Ca2+ was also unequivocally demonstrated by the exchange of slightly different small subunits between μ-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 Ca2+-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. 4-morpholineethanesulfonic acid The classical μ- and m-calpains are cytosolic Ca2+-dependent cysteine proteases that are ubiquitously expressed and differ in their sensitivity to Ca2+. They consist of an isoform-specific catalytic ∼80-kDa subunit (from the genes capn1 andcapn2, respectively) and a common regulatory ∼28-kDa subunit (capn4). Several other calpain-related genes are now known, but within this report, the word calpain refers only to the μ- and m-calpains. Whereas the exact physiological roles of calpains remain to be defined, many studies suggest that they have important cellular roles. They have been implicated in several important cellular functions, 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, 2Molinari M. Carafoli E. J. Membr. Biol. 1997; 156: 1-8Crossref PubMed Scopus (138) Google Scholar, 3Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (106) Google Scholar, 4Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar). Unlike many other cysteine proteases, calpains tend to cleave substrates at interdomain boundaries, thereby serving to modulate the function of these substrates rather than simply digesting them (5Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (140) Google Scholar). Several probable substrates have been identified both in vitro andin vivo including p53, protein kinase C, spectrin, Ca2+-ATPase, talin, and fibronectin (4Carafoli E. Molinari M. Biochem. Biophys. Res. Commun. 1998; 247: 193-203Crossref PubMed Scopus (340) Google Scholar, 5Suzuki K. Sorimachi H. FEBS Lett. 1998; 433: 1-4Crossref PubMed Scopus (140) Google Scholar, 6Kubbutat M.H. Vousden K.H. Mol. Cell. Biol. 1997; 17: 460-468Crossref PubMed Scopus (275) Google Scholar, 7Harada K. Maekawa T. Abu Shama K.M. Yamashima T. Yoshida K. J. Neurochem. 1999; 72: 2556-2564Crossref PubMed Scopus (40) Google Scholar, 8Dourdin N. Balcerzak D. Brustis J.J. Poussard S. Cottin P. Ducastaing A. Exp. Cell Res. 1999; 246: 433-442Crossref PubMed Scopus (54) Google Scholar). Both μ- and m-calpain are absolutely dependent on Ca2+for hydrolysis of their substrates (9Goll D.E. Kleese W.C. Okitani A. Kuwamoto T. Cong J. Karpell H.P. Mellgren R.L. Murachi T. Intracellular Calcium-dependent Proteolysis. CRC Press, Inc., Boca Raton, FL1990: 103-114Google Scholar, 10Sorimachi H. Saido T.C. Suzuki K. FEBS Lett. 1995; 343: 1-5Crossref Scopus (175) Google Scholar, 11Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar). The initial effects of Ca2+ binding to calpain include a conformational change that is essential to assemble the active site and some limited autolysis of both subunits. Further results of Ca2+ binding include aggregation of the whole enzyme or continued autolysis and degradation. The recent structure determination of m-calpain (12Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 13Strobl 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) has provided new insights into the structural basis of calpain activation by Ca2+. In the absence of Ca2+, the catalytic triad is not assembled so that the protease is inactive. Several structural features have been identified that maintain the active site in an inactive conformation. These involve on one side contacts between the large subunit N-terminal peptide of domain I and domain VI of the small subunit and on the other side contacts between domains II and III of the large subunit. Some of the later interactions have been clarified by mutational analysis (14Hosfield C.M. Moldoveanu T. Davies P.L. Elce J.S. Jia Z. J. Biol. Chem. 2001; 276: 7404-7407Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), but the functional implications of the binding of the N-terminal peptide to domain VI in relation to enzyme activation, autolysis, dissociation, and aggregation are not well characterized. Although there is a clear difference in the Ca2+requirement for activation (250–350 μm for m-calpain and 10–50 μm for μ-calpain), the basis for this difference remains elusive. However, the activation of both m- and μ-calpain appears to be very similar, and it was proposed that one function of Ca2+ was to cause the dissociation of the calpain subunits, a dissociation that may be reversible before autolysis but is promoted and presumably irreversible following autolysis (15Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. Biochem. Biophys. Res. Commun. 1995; 208: 376-383Crossref PubMed Scopus (80) Google Scholar, 16Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. FEBS Lett. 1995; 358: 101-103Crossref PubMed Scopus (82) Google Scholar, 17Kitagaki H. Tomioka S. Yoshizawa T. Sorimachi H. Saido T.C. Ishiura S. Suzuki K. Biosci. Biotechnol. Biochem. 2000; 64: 689-695Crossref PubMed Scopus (21) Google Scholar). In two other reports, however, subunit dissociation was not detected (18Zhang W. Mellgren R.L. Biochem. Biophys. Res. Commun. 1996; 227: 890-896Crossref Scopus (34) Google Scholar, 19Dutt P. Arthur J.S.C. Croall D.E. Elce J.S. FEBS Lett. 1998; 436: 367-371Crossref PubMed Scopus (24) Google Scholar). The inconsistent results stemmed from the complication of Ca2+-induced aggregation of calpain, and the difficulty in designing unambiguous experiments. In the presence of heavy precipitation, further studies of the calpain activation process will become extremely difficult. Consequently, several important aspects of the process of calpain activation and subsequent aggregation remain poorly understood. In our attempts to crystallize either wild-type or inactive m-calpain (C105S-m-80k/21k) in the presence of Ca2+, crystals were formed under certain conditions that contained only the small subunit together with an amorphous aggregate enriched in the large subunit. We therefore used light scattering and analytical ultracentrifugation to search for conditions in which Ca2+-induced aggregation and possibly dissociation could be avoided. Aggregation of calpain has frequently been noted, but the factors involved have not been systematically studied (15Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. Biochem. Biophys. Res. Commun. 1995; 208: 376-383Crossref PubMed Scopus (80) Google Scholar, 16Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. FEBS Lett. 1995; 358: 101-103Crossref PubMed Scopus (82) Google Scholar, 19Dutt P. Arthur J.S.C. Croall D.E. Elce J.S. FEBS Lett. 1998; 436: 367-371Crossref PubMed Scopus (24) Google Scholar, 20Elce J.S. Davies P.L. Hegadorn C. Maurice D.H. Arthur J.S.C. Biochem. J. 1997; 326: 31-38Crossref PubMed Scopus (46) Google Scholar). Subunit dissociation was also studied by the above methods and also by an improved subunit exchange experiment. The results confirmed unequivocally the occurrence of subunit dissociation in the presence of Ca2+ and provided some new insight into the nature of calpain aggregation. Both m-calpain and μ-calpain consist of an 80-kDa large subunit, which has an ∼62% sequence identity between the isoforms, and a 28-kDa small subunit, which is identical in the two isoforms. Upon Ca2+-induced activation, calpain undergoes autolysis in both subunits. The natural rat calpain small subunit contains 270 residues, of which the N-terminal glycine-rich region of ∼83 residues (domain V) is unstructured in the crystal structure (13Strobl 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) and is rapidly removed by autolysis (20Elce J.S. Davies P.L. Hegadorn C. Maurice D.H. Arthur J.S.C. Biochem. J. 1997; 326: 31-38Crossref PubMed Scopus (46) Google Scholar). In this work, our recombinant calpains contain either a 21-kDa small subunit of 184 residues (domain VI), corresponding closely to the natural small subunit autolysis product (20Elce J.S. Davies P.L. Hegadorn C. Maurice D.H. Arthur J.S.C. Biochem. J. 1997; 326: 31-38Crossref PubMed Scopus (46) Google Scholar, 21Elce J.S. Hegadorn C. Gauthier S. Vince J.W. Davies P.L. Protein Eng. 1995; 8: 843-848Crossref PubMed Scopus (65) Google Scholar), or an N-terminal His-tag version of this 21-kDa subunit containing 205 residues. The absence of domain V and the presence or absence of the N-terminal His-tag do not affect the stability or Ca2+requirement of the enzyme. In this work, inactive recombinant calpains were used to eliminate the problem of autolysis. C105S-m-80k-CHis6/21k m-calpain and the 21-kDa small subunit were prepared as described previously (21Elce J.S. Hegadorn C. Gauthier S. Vince J.W. Davies P.L. Protein Eng. 1995; 8: 843-848Crossref PubMed Scopus (65) Google Scholar) and stored in snap-frozen aliquots at a concentration of 10–20 mg/ml. A hybrid calpain large subunit was constructed by means of engineeredBamHI and DraI restriction sites. The resultant cDNA codes for m-calpain residues 1–48, μ-calpain residues 59–648, and m-calpain residues 637–714 (including the C-terminal His-tag). 1P. Dutt, and J. S. Elce, unpublished data. This large subunit formed an active calpain entitled m-Bam-μ-Dra-m-80k/21k and very similar to wild-type μ-calpain when coexpressed with the 21-kDa or NHis10-21-kDa small subunit. In its active form, m-Bam-μ-Dra-m-80k/21k is almost indistinguishable from μ-calpain on column chromatography and casein zymography, and its Ca2+ requirement of 120 μm is close to that of μ-calpain (∼25 μm) (m-calpain, 325 μm). The same hybrid calpain large subunit was also prepared with the inactivating mutation C115S. A recombinant form of rat calpastatin domain I with a C-terminal His-tag containing a total of 140 amino acid residues was also cloned, expressed, and purified as described previously (22Maki M. Hitomi K. Elce J.S. Calpain Methods and Protocols. Humana Press, NJ2000: 85-94Google Scholar). Crystallization experiments were carried out at room temperature by means of hanging drop/vapor diffusion. Samples of C105S-m-80k-CHis6/21k calpain (10–20 mg/ml) were preincubated in 50 mm Tris, pH 7.6, 0.2 mNaCl, 0.2 mm EDTA, 0.1% sodium azide, 10 mmdithiothreitol, and 10 mm CaCl2 at room temperature for 20 min. Incubation with 10 mmCaCl2 at a lower NaCl concentration caused immediate aggregation. A drop of this enzyme solution (2–5 μl) was mixed with an equal volume of reservoir solutions containing either (a) 1–2 m ammonium sulfate, 5–10% isopropyl alcohol, and one of several buffers including 50 mm sodium acetate, pH 5–5.5, 50 mmMES,2 pH 6.5, 50 mm HEPES, pH 7.5, and 50 mm Tris-HCl, pH 8.5, or (b) 20–25% polyethylene glycol 5000 monomethyl ether, 15–25% isopropyl alcohol, and 50 mm MES, pH 6.5. Crystals were obtained within 3–7 days in several conditions together with substantial amounts of amorphous aggregate, and the two solids could not be readily separated. The whole droplet was transferred to an Eppendorf tube and centrifuged. The clear supernatant (mother liquor) was removed, and the solid phase was washed repeatedly with crystallization buffer. The solid phase was then treated briefly with 20 μl of distilled water at room temperature, which instantly dissolved the crystals but did not significantly dissolve the amorphous precipitate. This residual precipitate was dissolved in SDS gel sample buffer, and the calpain subunit content of all the samples was analyzed by SDS gel electrophoresis. Light scattering by C105S-m-80k-CHis6/21k calpain was observed in a Perkin-Elmer LS50B spectrophotometer at room temperature. Both excitation and emission wavelengths were set at 320 nm, and the time-dependent change in scattering intensity was recorded. The solutions contained 70–200 μg/ml of calpain, 0–0.2m NaCl, 10 mm dithiothreitol, and 20–330 mm buffer, in a total volume of 3.0 ml. The buffer system was varied to alter the pH, and the CaCl2 concentration was varied from 0.1–50 mm. The volume of 3.0 ml used for conventional light scattering required too much material for studies at a higher protein concentration. Therefore, dynamic light-scattering experiments were performed at 20 °C using the DynaPro-MS/X instrument (Protein Solutions. Charlottesville, VA), which has an operating volume of 12 μl. For these experiments, protein solutions in the range of 1–5 mg/ml in 50 mm Tris-HCl, pH 7.6, containing 50 mm CaCl2 and 0.2–1.0m NaCl were used. The monodispersity or polydispersity of the solutions was assessed, and the molecular weight(s) of the predominant species were calculated. Sedimentation velocity experiments were carried out with m-calpain at 20 °C in a Beckman XL-I analytical ultracentrifuge using absorbance optics following the procedure described by Laue and Stafford (23Laue T.M. Stafford III, W.F. Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 75-100Crossref PubMed Scopus (236) Google Scholar). Protein solutions were exhaustively dialyzed against 50 mm Tris-HCl, pH 7.6, containing 200 mm NaCl, 2 mmtri-(2-carboxyethyl)-phosphine, and either 5 mm EDTA or 5 mm CaCl2. Runs were performed at a protein concentration of 1.13 mg/ml both in the presence and absence of Ca2+ at either 50,000 or 60,000 rpm for ∼3 h, during which time a minimum of 50 scans was taken to monitor the sedimentation rate of the protein. For sedimentation equilibrium, experiments were performed at three different protein concentrations of 0.09–0.3 mg/ml and at a minimum of two different speeds between 6000–12,000 rpm. Each speed was maintained until there was no significant difference inr 2/2 versus absorbance scans that were taken 2 h apart. To observe subunit interchange, the inactive mutants of μ- and m-type calpains, m-Bam-C115S-μ-Dra-m-80k/NHis10-21k and C105S-m-80k/21k, were dialyzed at 4 °C in buffer A (50 mm Tris-HCl, pH 7.6, 2 mm EDTA, 10 mm 2-mercaptoethanol, 0.1% (v/v) Triton X-100). Equal amounts (0.75 mg each) of the two proteins were mixed and resolved by chromatography on a Bio-Rad UnoQ (1 ml) ion exchange column with a gradient of [NaCl] in buffer A. An identical mixture at 20 °C was adjusted to 0.2 m NaCl, incubated with 5 mm net Ca2+ for 30 min, and then quenched with excess EDTA. The mixture was dialyzed against buffer A overnight at 4 °C and resolved on the UnoQ column as described above. The protein content of the fractions was analyzed by SDS gel electrophoresis, Coomassie Blue staining, and by immunoblotting. Following the preincubation of C105S-m-80kCHis6/21k in the presence of 10 mmCaCl2, clusters of needle-shaped crystals appeared in droplets containing either 50 mm sodium acetate, pH 5.0–5.5, or 50 mm MES, pH 6.5, in addition to 2m ammonium sulfate and 5% isopropyl alcohol. Crystals of the same morphology appeared, although less reproducibly in 50 mm MES, 1.6 m ammonium sulfate, 10% isopropyl alcohol. In a different experiment, very thin hexagonal plate crystals were obtained from 100 mm MES, pH 6.5, 20% polyethylene glycol 5000 monomethyl ether, and 20% isopropyl alcohol. Under all these conditions, no crystals were obtained from calpain that had not been preincubated with CaCl2. Crystals of either morphology diffracted x-rays to ∼3.0 Å at a rotating anode x-ray source. However, further crystallographic characterization was not possible because of twin crystal formation. SDS gel electrophoresis showed that the crystals obtained from these trials contained only the 21-kDa small subunit. The mother liquor showed faint traces of the 21-kDa protein, and the amorphous precipitate contained the 80-kDa large subunit together with a very small amount of the 21-kDa protein (Fig. 1). At low ionic strength, calpain aggregated on the addition of sufficient Ca2+, and this could be reversed rapidly and completely by excess EDTA and more slowly reversed by raising the NaCl concentration (Figs. 2, a and b). In buffers containing 70 μg/ml calpain and 0.2 m NaCl, no detectable increase in light scattering was caused by the addition of Ca2+. Aggregation at low ionic strength was also prevented by the presence of calpastatin, and in this case, the small increase in light scattering was assumed to mark the onset of the calpastatin-calpain complex formation (Fig. 2 c). The calpastatin used here was a recombinant form of rat calpastatin domain I containing a total of 140 residues (22Maki M. Hitomi K. Elce J.S. Calpain Methods and Protocols. Humana Press, NJ2000: 85-94Google Scholar). Table I lists the Ca2+requirements for these two different events and shows that aggregation of C105S-m-80k/21k calpain was dependent on the ionic strength and pH of the buffer as well as on the Ca2+ concentration. The Ca2+ concentration required in any given buffer for calpastatin-calpain complex formation was significantly higher than that required for aggregation (24Kapprell H.P. Goll D.E. J. Biol. Chem. 1989; 264: 17888-17896Abstract Full Text PDF PubMed Google Scholar).Table ICa 2+ requirement for calpain aggregation and for calpastatin-calpain complex formation under different experimental conditionsBuffer conditionsCa2+ requirement for calpain aggregationCa2+ requirement for calpastatin-calpain complex formationmmmm0.1 m MES, pH 6.250.20.3–0.50.1 m MES, pH 6.25, 0.1 m NaClnono0.33 m MES, pH 6.25nono0.1 m HEPES, pH 7.01–2320 mm HEPES, pH 7.50.82–30.1m HEPES, pH 7.53520 mm Tris-HCl, pH 7.61–25–6100 mm Tris-HCl, pH 7.6nonoThe calpain concentration was constant at 70 μg/ml (0.7 μm), and calpastatin (a recombinant form of rat calpastatin domain I of 140 residues) was added at a concentration of 1.4 μm. The Ca2+ requirement was estimated from the point at which an abrupt change in light scattering occurred during stepwise addition of Ca2+. The experiments were carried out at 20 °C. Open table in a new tab The calpain concentration was constant at 70 μg/ml (0.7 μm), and calpastatin (a recombinant form of rat calpastatin domain I of 140 residues) was added at a concentration of 1.4 μm. The Ca2+ requirement was estimated from the point at which an abrupt change in light scattering occurred during stepwise addition of Ca2+. The experiments were carried out at 20 °C. Dynamic light-scattering experiments were conducted at higher calpain concentrations than the light-scattering work in order to approach conditions relevant to crystallization trials. They showed that a solution of 1 mg/ml of m-calpain in 50 mm Tris-HCl, pH 7.6, was monodisperse in the absence of Ca2+. The major species had a molecular mass of 95–105 kDa, and ∼5% of the protein was present as high molecular mass species in the range of at least 103 kDa. At this low ionic strength, the solution became polydisperse upon the addition of Ca2+ but remained monodisperse in 0.5 m NaCl with Ca2+. With 3–5 mg/ml calpain in 1 m NaCl in the presence of Ca2+, 50–70% of the scattering intensities were contributed by the monomer of ∼100 kDa, and the remainder were provided by components of the order of 103-105kDa. The data did not indicate the presence of the homodimer of 21 kDa. Sedimentation studies also provided clear evidence of aggregation. Protein samples (1.13 mg/ml) gave rise to a sedimentation coefficient value ( s20,w0) of 5.93 in the absence of Ca2+, whereas in the presence of Ca2+ the coefficient value was 17.79. In the sedimentation equilibrium experiment, m-calpain (0.09–0.3 mg/ml) in the absence of Ca2+ showed a single molecular species of 100 kDa, but in the presence of Ca2+, multiple species were observed with molecular masses ranging from 276 to 600 kDa, suggesting the presence of a mixture of aggregates of 4–8 calpain molecules or calpain large subunits. The data again did not provide evidence for the existence of the isolated small subunit. As a control, the isolated 21-kDa small subunit protein, which is known to exist as a homodimer (25Blanchard H. Grochulski P. Li Y. Arthur J.S.C. Davies P.L. Elce J.S. Cygler M. Nat. Struct. Biol. 1997; 4: 532-538Crossref PubMed Scopus (182) Google Scholar), was analyzed at the same time under the same conditions. In the absence of Ca2+, it was monodisperse with an apparent molecular mass of 40 kDa. In the presence of Ca2+, it was polydisperse with a dominant species of an apparent molecular mass of 47.5 kDa. The subunit exchange experiment depended on two factors, the separation by ion exchange chromatography of the μ-calpain-like m-Bam-C115S-μ-Dra-m-80k/NHis10-21k calpain from the m-type C105S-m-80k/21k calpain and the clear distinction on gel electrophoresis between the two different small subunits with and without the NHis10 peptide. Buffers containing 0.2 m NaCl and 0.1% Triton X-100 were used for incubation with Ca2+ to minimize the formation of insoluble aggregates. The chromatograms (Fig. 3) show that μ- and m-calpain peaks, which were well separated before exposure to Ca2+, were recovered in the same positions after a 30-min exposure to Ca2+ followed by quenching in EDTA and extensive dialysis. The peaks were less sharp following Ca2+ treatment, and the yield of protein was ∼60%, suggesting some degree of aggregation and some failure to reform heterodimers. The Coomassie Blue-stained gel (data not shown) and the immunoblot (Fig. 4) showed clearly that some NHis10-21k small subunit had been transferred from the μ-type calpain to the m-calpain, and conversely that some 21-kDa small subunit had been transferred from the m-calpain to the μ-type calpain. The conclusion is inescapable that in these inactive calpains, which cannot undergo autolysis, subunit interchange had occurred so that some large and small subunits must have dissociated in the presence of Ca2+ and at least to some extent reversibly reassociated upon the removal of Ca2+. Densitometry was not performed, but the extent of exchange appears from the immunoblot to be of the order of 30%. The nature of this experiment with a column step of relatively low recovery does not permit precise quantification of the extent of subunit exchange.Figure 4Immunoblot analysis of the eluted peaks shown in Fig. 3. Fractions spanning the two main eluted peaks in each column were analyzed by means of immunoblotting. Theleft-hand section of the blot contains samples from the column (see Fig. 3 a) previous to Ca2+ treatment, and the right-hand section contains samples from the column (see Fig. 3 b) following exposure to Ca2+. Theupper portion of the blot was treated with a mixture of a polyclonal antibody to rat m-calpain large subunit and a monoclonal antibody to human μ-calpain large subunit, which cross-reacts with rat μ-calpain. The lower portion of the blot was treated with a polyclonal antibody to the rat calpain small subunit. Both blots were treated with appropriate second antibodies and then developed by enhanced chemiluminescence. It is important to note the appearance of the NHis1021k subunit in the m-calpain peak and the corresponding appearance of the 2kDa subunit in the μ-calpain peak only after Ca2+ treatment as indicated by open arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The impetus for this work was the need to crystallize calpain in the presence of Ca2+. The structure of calpain in the absence of Ca2+ (12Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 13Strobl 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) showed that the active site was not assembled to a catalytically active conformation, so that it was clearly of interest to solve the structure in the presence of Ca2+ to understand the mechanism of calpain activation. Not unexpectedly, however, crystallization in the presence of Ca2+ raised problems of aggregation and subunit dissociation. The suggestion that the calpain subunits dissociate in the presence of Ca2+ has been the subject of some debate (15Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. Biochem. Biophys. Res. Commun. 1995; 208: 376-383Crossref PubMed Scopus (80) Google Scholar, 16Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. FEBS Lett. 1995; 358: 101-103Crossref PubMed Scopus (82) Google Scholar, 17Kitagaki H. Tomioka S. Yoshizawa T. Sorimachi H. Saido T.C. Ishiura S. Suzuki K. Biosci. Biotechnol. Biochem. 2000; 64: 689-695Crossref PubMed Scopus (21) Google Scholar, 18Zhang W. Mellgren R.L. Biochem. Biophys. Res. Commun. 1996; 227: 890-896Crossref Scopus (34) Google Scholar, 19Dutt P. Arthur J.S.C. Croall D.E. Elce J.S. FEBS Lett. 1998; 436: 367-371Crossref PubMed Scopus (24) Google Scholar) arising not least from the difficulty of designing definitive experiments. Here we provide evidence that clearly confirms the phenomenon of subunit dissociation first described by Yoshizawaet al. (15Yoshizawa T. Sorimachi H. Tomioka S. Ishiura S. Suzuki K. Biochem. Biophys. Res. Commun. 1995; 208: 376-383Crossref PubMed Scopus (80) Google Scholar) in 1995. The results also suggest that in m-calpain and in the absence of autolysis, the aggregation/dissociation is a highly reversible process. The dissociation is normally not complete unless driven by other factors. Extrapolation of these clearin vitro results to the cell is necessarily speculative. It seems highly probable that the subunit dissociation is an essential aspect of calpain activation in vivo, but the ensuing aggregation observed in vitro is less likely to be relevantin vivo where calpain is diluted in a highly proteinaceous environment and where autolysis rapidly removes the activated calpain. Calpain aggregation is clearly a function of protein concentration, ionic strength, and Ca2+ concentration, but some new insights into the aggregation were obtained from light-scattering and ultracentrifugation experiments. First, within the experimental time frame (∼1 h) and in the absence of autolysis, aggregation is an equilibrium process, because it could be fully reversed by the addition of EDTA. Second, the inhibition of aggregation by higher salt concentration strongly suggests that hydrophobic interactions are involved. Although aggregation of calpain at <200 μg/ml appeared to be suppressed by 0.2 m NaCl, both dynamic light scattering and ultracentrifugation run at ≥ 1-mg/ml calpain showed that soluble high molecular weight oligomers were formed in the presence of Ca2+, which could only partly be suppressed by 0.5 or 1m NaCl. Both of these methods failed to detect the presence of the isolated 21-kDa homodimer, which was anticipated as a result of subunit dissociation. Previous work on the 21-kDa subunit showed that it exists as a homodimer both in the presence and absence of Ca2+, and that the homodimer once formed is unlikely to dissociate in any of the conditions used here (25Blanchard H. Grochulski P. Li Y. Arthur J.S.C. Davies P.L. Elce J.S. Cygler M. Nat. Struct. Biol. 1997; 4: 532-538Crossref PubMed Scopus (182) Google Scholar). The absence of free small subunits in the light-scattering and centrifugation experiments therefore suggests that subunit dissociation is far from complete in these conditions, and that the large soluble complexes are still composed predominantly of calpain (80 + 21 kDa) heterodimers, which have undergone Ca2+-induced conformational changes leading to aggregation. It is possible to imagine a highly flexible intermediate form of the heterodimer in which the contact between the N-terminal peptide of the large subunit and domain VI in the small subunit has been lost, whereas the subunits remain at least partially attached through residual contacts between domains IV and VI. A third aspect of the aggregation studies was the inhibition of aggregation by a molar excess of a 140-residue domain of calpastatin. Separate sections of calpastatin are known to bind to domain IV in the large subunit and to domain VI of the small subunit (26Takano E. Ma H. Yang H.Q. Maki M. Hatanaka M. FEBS Lett. 1995; 362: 93-97Crossref PubMed Scopus (73) Google Scholar). Therefore, it seems probable that calpastatin prevents calpain aggregation by binding to both subunits simultaneously and preventing subunit dissociation. Whereas m-calpain dissociation appears to be incomplete in many buffer conditions, it is clear that calpain does indeed dissociate in the presence of Ca2+ at pH 6.5 in the conditions prevailing in some crystallization droplets. In these precipitant conditions, the dissociated 21-kDa small subunit crystallized out, and its removal presumably promoted further subunit dissociation, leaving the large subunit to precipitate as an amorphous aggregate. Several crystallization conditions have also been found in which no visible aggregation of calpain occurs in the presence of Ca2+, but useful crystals, whether they are of the activated heterodimer or of the isolated large subunit, have not yet been obtained. Finally, the subunit exchange experiment also clearly demonstrated reversible subunit dissociation of calpain, because the interchange of small subunits between the m- and μ-type is only possible provided they dissociate. We had earlier failed to detect subunit dissociation by means of column chromatography of calpain in the presence of Ca2+ and by a less definitive version of the subunit exchange experiment (19Dutt P. Arthur J.S.C. Croall D.E. Elce J.S. FEBS Lett. 1998; 436: 367-371Crossref PubMed Scopus (24) Google Scholar), but the present experiments provide unequivocal proof of subunit dissociation. The importance of hydrophobic interactions and the effects of calpastatin support the idea that aggregation is a result of subunit dissociation. The crystal structure of calpain shows that the large and small subunits in the absence of Ca2+ are bound mainly by interactions between the fifth EF-hand motif of each subunit (12Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 13Strobl 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 a manner very similar to that shown for the 21-kDa homodimer (25Blanchard H. Grochulski P. Li Y. Arthur J.S.C. Davies P.L. Elce J.S. Cygler M. Nat. Struct. Biol. 1997; 4: 532-538Crossref PubMed Scopus (182) Google Scholar,27Lin G.D. Chattopadhyay D. Maki M. Wang K.K. Carson M. Jin L. Yuen P.W. Takano E. Hatanaka M. DeLucas L.J. Nat. Struct. Biol. 1997; 4: 539-547Crossref PubMed Scopus (178) Google Scholar). Dissociation must expose the complementary dimerization interface on both subunits, which is a large hydrophobic area of ∼2780 Å2 representing approximately one quarter of the surface area of domain IV and equally of domain VI (Fig. 5). Such an exposure is energetically unfavorable in aqueous solution and would be expected to promote either "correct" reassociation of the large and small subunits or random association leading to aggregation. The exposure of this surface would be even more disfavored in solutions of higher ionic strength, which explains the partial suppression of calpain aggregation in 0.2–1m NaCl. Our modeling suggests that two large subunits could not dimerize at this site in domain IV in their correct relative orientation as observed in the domain VI homodimer because of steric interference by domains I–III. Thus, even partial exposure of this hydrophobic patch would promote the formation of randomly associated aggregates. Based on the first x-ray structure of m-calpain (12Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar), we proposed that the constraints imposed upon the protease domains I and II by the remainder of the molecule would act as a barrier in the activation of calpain. These constraints were provided on the one side by the small subunit domain VI, binding to the large subunit N-terminal peptide of domain I, and on the other side by a set of salt links between domains II and III. The release of the constraints would facilitate the assembly of the competent catalytic triad. We have reported experiments supporting the idea of the domain II/domain III interaction (14Hosfield C.M. Moldoveanu T. Davies P.L. Elce J.S. Jia Z. J. Biol. Chem. 2001; 276: 7404-7407Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and this work suggests that dissociation of the small subunit would release the constraint on domain I. The evidence of aggregation, calpastatin complex formation, and subunit dissociation suggests that subunit dissociation is the principal cause of Ca2+-induced aggregation of calpain. Furthermore, we also show that both dissociation and aggregation are reversible. Our present understanding of these events is shown in Fig. 5. Ca2+-induced conformational rearrangement and partial dissociation leads to the formation of aggregates probably containing heterodimers as well as dissociated large subunits. This step is largely reversible, but the small subunit homodimer is probably no longer available for reassociation, and larger aggregates composed almost exclusively of large subunit will be formed almost irreversibly. In the case of active calpains, aggregation also occurs in vitro on initial exposure to Ca2+, but autolysis at the same time begins to degrade the large subunits to inactive fragments. We thank L. Hicks and co-workers at the Alberta Peptide Institute, University of Alberta, for carrying out the ultracentrifugation experiments and for advice on interpretation. We also thank Dr. C. M. Hosfield for discussions and Teresa DeVeyra for skilled technical assistance.
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