PDBsum entry 1df0

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Hydrolase PDB id
Protein chains
624 a.a. *
176 a.a. *
Waters ×358
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Crystal structure of m-calpain
Structure: M-calpain. Chain: a. Fragment: large (catalytic) subunit. Synonym: calcium-activated neutral proteinase, calpain 2. Engineered: yes. Mutation: yes. Calpain. Chain: b. Fragment: domain vi (calcium-binding domain), small (regula
Source: Rattus norvegicus. Norway rat. Organism_taxid: 10116. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562
Biol. unit: Dimer (from PQS)
2.60Å     R-factor:   0.223     R-free:   0.293
Authors: C.M.Hosfield,J.S.Elce,P.L.Davies,Z.Jia
Key ref:
C.M.Hosfield et al. (1999). Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J, 18, 6880-6889. PubMed id: 10601010 DOI: 10.1093/emboj/18.24.6880
16-Nov-99     Release date:   21-Jun-00    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q07009  (CAN2_RAT) -  Calpain-2 catalytic subunit
700 a.a.
624 a.a.*
Protein chain
Pfam   ArchSchema ?
Q64537  (CPNS1_RAT) -  Calpain small subunit 1
270 a.a.
176 a.a.
Key:    PfamA domain  PfamB domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 1 residue position (black cross)

 Enzyme reactions 
   Enzyme class: Chain A: E.C.  - Calpain-2.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Cofactor: Calcium
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     intracellular   9 terms 
  Biological process     cellular response to amino acid stimulus   6 terms 
  Biochemical function     protein binding     9 terms  


DOI no: 10.1093/emboj/18.24.6880 EMBO J 18:6880-6889 (1999)
PubMed id: 10601010  
Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation.
C.M.Hosfield, J.S.Elce, P.L.Davies, Z.Jia.
The combination of thiol protease activity and calmodulin-like EF-hands is a feature unique to the calpains. The regulatory mechanisms governing calpain activity are complex, and the nature of the Ca(2+)-induced switch between inactive and active forms has remained elusive in the absence of structural information. We describe here the 2.6 A crystal structure of m-calpain in the Ca(2+)-free form, which illustrates the structural basis for the inactivity of calpain in the absence of Ca(2+). It also reveals an unusual thiol protease fold, which is associated with Ca(2+)-binding domains through heterodimerization and a C(2)-like beta-sandwich domain. Strikingly, the structure shows that the catalytic triad is not assembled, indicating that Ca(2+)-binding must induce conformational changes that re-orient the protease domains to form a functional active site. The alpha-helical N-terminal anchor of the catalytic subunit does not occupy the active site but inhibits its assembly and regulates Ca(2+)-sensitivity through association with the regulatory subunit. This Ca(2+)-dependent activation mechanism is clearly distinct from those of classical proteases.
  Selected figure(s)  
Figure 3.
Figure 3 Calpain has a unique N-terminal anchor. (A) The helical anchor (residues 2 -16 are shown) makes contacts only with D-VI (colors as in Figure 1). (B) View down the helical axis highlights interactions between the residues in the anchor (magenta type) and D-VI (black type), represented as an electrostatic GRASP surface (Nicholls et al., 1991) (red, acidic; blue, basic). (C) Side view of (B) illustrates the depth of the hydrophobic pocket in D-VI, which interacts with hydrophobic residues Ala2, Gly3, Ile4, Ala5, Leu8 and Ala9 of the anchor. This anchor inhibits active site assembly by associating with the regulatory subunit, thus restricting flexibility of protease D-I. The anchor also acts as a co-chaperone in concert with D-VI, ensuring proper folding of the catalytic subunit. (B) and (C) were created with the program GRASP (Nicholls et al., 1991).
Figure 4.
Figure 4 Domain III shares similar characteristics with a C[2] domain. A typical C[2] domain exists as an anti-parallel -sandwich with several acidic residues at one end that form a binding cradle for Ca^2+. The first C[2] domain from synaptotagmin (cyan, PDB accession code 1RSY) (Sutton et al., 1995) and D-III (green) have approximately the same overall dimensions, though slightly differing topologies. Numerous acidic residues (red) result in a highly negative potential, which is partially stabilized by adjacent basic residues (blue).
  The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO J (1999, 18, 6880-6889) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21508973 S.J.Storr, N.O.Carragher, M.C.Frame, T.Parr, and S.G.Martin (2011).
The calpain system and cancer.
  Nat Rev Cancer, 11, 364-374.  
18073773 M.B.Bevers, and R.W.Neumar (2008).
Mechanistic role of calpains in postischemic neurodegeneration.
  J Cereb Blood Flow Metab, 28, 655-673.  
19020623 R.A.Hanna, R.L.Campbell, and P.L.Davies (2008).
Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin.
  Nature, 456, 409-412.
PDB code: 3bow
19020622 T.Moldoveanu, K.Gehring, and D.R.Green (2008).
Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.
  Nature, 456, 404-408.
PDB code: 3df0
17908326 A.J.Scotter, M.Guo, M.M.Tomczak, M.E.Daley, R.L.Campbell, R.J.Oko, D.A.Bateman, A.Chakrabartty, B.D.Sykes, and P.L.Davies (2007).
Metal ion-dependent, reversible, protein filament formation by designed beta-roll polypeptides.
  BMC Struct Biol, 7, 63.  
17608959 D.E.Croall, and K.Ersfeld (2007).
The calpains: modular designs and functional diversity.
  Genome Biol, 8, 218.  
17429681 D.L.Enns, T.Raastad, I.Ugelstad, and A.N.Belcastro (2007).
Calpain/calpastatin activities and substrate depletion patterns during hindlimb unweighting and reweighting in skeletal muscle.
  Eur J Appl Physiol, 100, 445-455.  
17210638 K.Tonami, Y.Kurihara, H.Aburatani, Y.Uchijima, T.Asano, and H.Kurihara (2007).
Calpain 6 is involved in microtubule stabilization and cytoskeletal organization.
  Mol Cell Biol, 27, 2548-2561.  
17416893 M.M.Peñas, A.Hervás-Aguilar, T.Múnera-Huertas, E.Reoyo, M.A.Peñalva, H.N.Arst, and J.Tilburn (2007).
Further characterization of the signaling proteolysis step in the Aspergillus nidulans pH signal transduction pathway.
  Eukaryot Cell, 6, 960-970.  
17942392 R.L.Mellgren, and X.Huang (2007).
Fetuin A stabilizes m-calpain and facilitates plasma membrane repair.
  J Biol Chem, 282, 35868-35877.  
17646163 S.Hata, N.Doi, F.Kitamura, and H.Sorimachi (2007).
Stomach-specific calpain, nCL-2/calpain 8, is active without calpain regulatory subunit and oligomerizes through C2-like domains.
  J Biol Chem, 282, 27847-27856.  
17640275 S.Vernarecci, G.Colotti, P.Ornaghi, E.Schiebel, E.Chiancone, and P.Filetici (2007).
The yeast penta-EF protein Pef1p is involved in cation-dependent budding and cell polarization.
  Mol Microbiol, 65, 1122-1138.  
16803906 E.Melloni, M.Averna, R.Stifanese, R.De Tullio, E.Defranchi, F.Salamino, and S.Pontremoli (2006).
Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?
  J Biol Chem, 281, 24945-24954.  
16476741 S.Hata, S.Koyama, H.Kawahara, N.Doi, T.Maeda, N.Toyama-Sorimachi, K.Abe, K.Suzuki, and H.Sorimachi (2006).
Stomach-specific calpain, nCL-2, localizes in mucus cells and proteolyzes the beta-subunit of coatomer complex, beta-COP.
  J Biol Chem, 281, 11214-11224.  
16216885 D.Cuerrier, T.Moldoveanu, and P.L.Davies (2005).
Determination of peptide substrate specificity for mu-calpain by a peptide library-based approach: the importance of primed side interactions.
  J Biol Chem, 280, 40632-40641.  
15691848 J.Takano, M.Tomioka, S.Tsubuki, M.Higuchi, N.Iwata, S.Itohara, M.Maki, and T.C.Saido (2005).
Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice.
  J Biol Chem, 280, 16175-16184.  
15653743 S.R.Dennison, S.Dante, T.Hauss, K.Brandenburg, F.Harris, and D.A.Phoenix (2005).
Investigations into the membrane interactions of m-calpain domain V.
  Biophys J, 88, 3008-3017.  
14976200 A.Alexa, Z.Bozóky, A.Farkas, P.Tompa, and P.Friedrich (2004).
Contribution of distinct structural elements to activation of calpain by Ca2+ ions.
  J Biol Chem, 279, 20118-20126.  
14993287 A.Glading, R.J.Bodnar, I.J.Reynolds, H.Shiraha, L.Satish, D.A.Potter, H.C.Blair, and A.Wells (2004).
Epidermal growth factor activates m-calpain (calpain II), at least in part, by extracellular signal-regulated kinase-mediated phosphorylation.
  Mol Cell Biol, 24, 2499-2512.  
15119965 A.Via, and M.Helmer-Citterich (2004).
A structural study for the optimisation of functional motifs encoded in protein sequences.
  BMC Bioinformatics, 5, 50.  
14982937 B.Bánfi, F.Tirone, I.Durussel, J.Knisz, P.Moskwa, G.Z.Molnár, K.H.Krause, and J.A.Cox (2004).
Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5).
  J Biol Chem, 279, 18583-18591.  
15073171 B.G.Diaz, T.Moldoveanu, M.J.Kuiper, R.L.Campbell, and P.L.Davies (2004).
Insertion sequence 1 of muscle-specific calpain, p94, acts as an internal propeptide.
  J Biol Chem, 279, 27656-27666.  
14749380 T.Franz, L.Winckler, T.Boehm, and T.N.Dear (2004).
Capn5 is expressed in a subset of T cells and is dispensable for development.
  Mol Cell Biol, 24, 1649-1654.  
14581465 T.Moldoveanu, Z.Jia, and P.L.Davies (2004).
Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites.
  J Biol Chem, 279, 6106-6114.  
12824178 C.Wang, J.K.Barry, Z.Min, G.Tordsen, A.G.Rao, and O.A.Olsen (2003).
The calpain domain of the maize DEK1 protein contains the conserved catalytic triad and functions as a cysteine proteinase.
  J Biol Chem, 278, 34467-34474.  
12857354 D.Mitchell, and A.Bell (2003).
PEST sequences in the malaria parasite Plasmodium falciparum: a genomic study.
  Malar J, 2, 16.  
12554956 G.P.Pal, T.DeVeyra, J.S.Elce, and Z.Jia (2003).
Purification, crystallization and preliminary X-ray analysis of a mu-like calpain.
  Acta Crystallogr D Biol Crystallogr, 59, 369-371.  
12885783 J.N.Henderson, J.Zhang, B.W.Evans, and K.Redding (2003).
Disassembly and degradation of photosystem I in an in vitro system are multievent, metal-dependent processes.
  J Biol Chem, 278, 39978-39986.  
12692242 M.Dimitrova, I.Imbert, M.P.Kieny, and C.Schuster (2003).
Protein-protein interactions between hepatitis C virus nonstructural proteins.
  J Virol, 77, 5401-5414.  
12711611 M.Mella, G.Colotti, C.Zamparelli, D.Verzili, A.Ilari, and E.Chiancone (2003).
Information transfer in the penta-EF-hand protein sorcin does not operate via the canonical structural/functional pairing. A study with site-specific mutants.
  J Biol Chem, 278, 24921-24928.  
12783889 O.Kifor, I.Kifor, F.D.Moore, R.R.Butters, and E.M.Brown (2003).
m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR.
  J Biol Chem, 278, 31167-31176.  
12500971 R.Betts, S.Weinsheimer, G.E.Blouse, and J.Anagli (2003).
Structural determinants of the calpain inhibitory activity of calpastatin peptide B27-WT.
  J Biol Chem, 278, 7800-7809.  
12591934 S.Gil-Parrado, O.Popp, T.A.Knoch, S.Zahler, F.Bestvater, M.Felgenträger, A.Holloschi, A.Fernández-Montalván, E.A.Auerswald, H.Fritz, P.Fuentes-Prior, W.Machleidt, and E.Spiess (2003).
Subcellular localization and in vivo subunit interactions of ubiquitous mu-calpain.
  J Biol Chem, 278, 16336-16346.  
12665854 T.Moldoveanu, C.M.Hosfield, D.Lim, Z.Jia, and P.L.Davies (2003).
Calpain silencing by a reversible intrinsic mechanism.
  Nat Struct Biol, 10, 371-378.
PDB code: 1mdw
14500891 Z.Mucsi, F.Hudecz, M.Hollósi, P.Tompa, and P.Friedrich (2003).
Binding-induced folding transitions in calpastatin subdomains A and C.
  Protein Sci, 12, 2327-2336.  
11830654 E.Carafoli (2002).
Calcium signaling: a tale for all seasons.
  Proc Natl Acad Sci U S A, 99, 1115-1122.  
12189137 E.Dainese, R.Minafra, A.Sabatucci, P.Vachette, E.Melloni, and I.Cozzani (2002).
Conformational changes of calpain from human erythrocytes in the presence of Ca2+.
  J Biol Chem, 277, 40296-40301.  
11784720 H.Patzke, and L.H.Tsai (2002).
Calpain-mediated cleavage of the cyclin-dependent kinase-5 activator p39 to p29.
  J Biol Chem, 277, 8054-8060.  
11909964 H.Shiraha, A.Glading, J.Chou, Z.Jia, and A.Wells (2002).
Activation of m-calpain (calpain II) by epidermal growth factor is limited by protein kinase A phosphorylation of m-calpain.
  Mol Cell Biol, 22, 2716-2727.  
12423339 K.Brandenburg, F.Harris, S.Dennison, U.Seydel, and D.Phoenix (2002).
Domain V of m-calpain shows the potential to form an oblique-orientated alpha-helix, which may modulate the enzyme's activity via interactions with anionic lipid.
  Eur J Biochem, 269, 5414-5422.  
11809743 P.Tompa, Z.Mucsi, G.Orosz, and P.Friedrich (2002).
Calpastatin subdomains A and C are activators of calpain.
  J Biol Chem, 277, 9022-9026.  
11814340 T.R.Pray, K.K.Reiling, B.G.Demirjian, and C.S.Craik (2002).
Conformational change coupling the dimerization and activation of KSHV protease.
  Biochemistry, 41, 1474-1482.  
11489944 A.Ruiz-Vela, F.Serrano, M.A.González, J.L.Abad, A.Bernad, M.Maki, and C.Martínez-A (2001).
Transplanted long-term cultured pre-BI cells expressing calpastatin are resistant to B cell receptor-induced apoptosis.
  J Exp Med, 194, 247-254.  
11717497 J.Jia, N.Borregaard, K.Lollike, and M.Cygler (2001).
Structure of Ca(2+)-loaded human grancalcin.
  Acta Crystallogr D Biol Crystallogr, 57, 1843-1849.
PDB codes: 1k94 1k95
11485997 M.Averna, R.De Tullio, F.Salamino, R.Minafra, S.Pontremoli, and E.Melloni (2001).
Age-dependent degradation of calpastatin in kidney of hypertensive rats.
  J Biol Chem, 276, 38426-38432.  
11238954 M.Azam, S.S.Andrabi, K.E.Sahr, L.Kamath, A.Kuliopulos, and A.H.Chishti (2001).
Disruption of the mouse mu-calpain gene reveals an essential role in platelet function.
  Mol Cell Biol, 21, 2213-2220.  
10825211 J.S.Arthur, J.S.Elce, C.Hegadorn, K.Williams, and P.A.Greer (2000).
Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division.
  Mol Cell Biol, 20, 4474-4481.  
  10783162 S.B.Sokol, and P.E.Kuwabara (2000).
Proteolysis in Caenorhabditis elegans sex determination: cleavage of TRA-2A by TRA-3.
  Genes Dev, 14, 901-906.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.