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- PDB-3df0: Calcium-dependent complex between m-calpain and calpastatin -

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Basic information

Entry
Database: PDB / ID: 3df0
TitleCalcium-dependent complex between m-calpain and calpastatin
Components
  • Calpain small subunit 1
  • Calpain-2 catalytic subunit
  • Calpastatin
KeywordsHYDROLASE / protease core domain / penta EF-hand domains / C2-like domain / inhibitor loop-out / Membrane / Protease / Thiol protease / Phosphoprotein / Protease inhibitor / Thiol protease inhibitor
Function / homology
Function and homology information


egg activation / calcium-dependent cysteine-type endopeptidase inhibitor activity / calpain-2 / negative regulation of type B pancreatic cell apoptotic process / Degradation of the extracellular matrix / positive regulation of phosphatidylcholine biosynthetic process / calpain complex / skeletal muscle atrophy / protein catabolic process at postsynapse / calcium-dependent cysteine-type endopeptidase activity ...egg activation / calcium-dependent cysteine-type endopeptidase inhibitor activity / calpain-2 / negative regulation of type B pancreatic cell apoptotic process / Degradation of the extracellular matrix / positive regulation of phosphatidylcholine biosynthetic process / calpain complex / skeletal muscle atrophy / protein catabolic process at postsynapse / calcium-dependent cysteine-type endopeptidase activity / perinuclear endoplasmic reticulum / myoblast fusion / myoblast differentiation / regulation of interleukin-6 production / positive regulation of myoblast fusion / positive regulation of cardiac muscle cell apoptotic process / regulation of protein catabolic process / behavioral response to pain / pseudopodium / protein autoprocessing / blastocyst development / synaptic vesicle endocytosis / animal organ regeneration / cellular response to interferon-beta / response to mechanical stimulus / cytoskeletal protein binding / liver development / proteolysis involved in protein catabolic process / cell projection / female pregnancy / cellular response to amino acid stimulus / protein catabolic process / response to hydrogen peroxide / presynapse / peptidase activity / cellular response to lipopolysaccharide / protease binding / postsynapse / lysosome / postsynaptic density / response to hypoxia / membrane raft / external side of plasma membrane / focal adhesion / neuronal cell body / dendrite / calcium ion binding / protein-containing complex binding / chromatin / Golgi apparatus / enzyme binding / endoplasmic reticulum / proteolysis / membrane / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Proteinase inhibitor I27, calpastatin / Calpastatin / Calpain inhibitor / CAPN2, penta-EF hand, calcium binding motif / Calpain subdomain III / : / Jelly Rolls - #380 / Peptidase C2, calpain, domain III / calpain_III / Peptidase C2, calpain, large subunit, domain III ...Proteinase inhibitor I27, calpastatin / Calpastatin / Calpain inhibitor / CAPN2, penta-EF hand, calcium binding motif / Calpain subdomain III / : / Jelly Rolls - #380 / Peptidase C2, calpain, domain III / calpain_III / Peptidase C2, calpain, large subunit, domain III / Peptidase C2, calpain family / Calpain large subunit, domain III superfamily / Calpain large subunit, domain III / Peptidase C2, calpain, catalytic domain / Calpain family cysteine protease / Cysteine proteinase, calpain-type, catalytic domain profile. / Calpain-like thiol protease family. / EF-hand domain pair / Cysteine proteinases / Cysteine peptidase, cysteine active site / Eukaryotic thiol (cysteine) proteases cysteine active site. / Cathepsin B; Chain A / EF-hand / Recoverin; domain 1 / Papain-like cysteine peptidase superfamily / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair / Jelly Rolls / Alpha-Beta Complex / Sandwich / Orthogonal Bundle / Mainly Beta / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Calpastatin / Calpain-2 catalytic subunit / Calpain small subunit 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.95 Å
AuthorsMoldoveanu, T. / Gehring, K. / Green, D.R.
Citation
Journal: Nature / Year: 2008
Title: Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains.
Authors: Moldoveanu, T. / Gehring, K. / Green, D.R.
#1: Journal: Cell(Cambridge,Mass.) / Year: 2002
Title: A Ca(2+) switch aligns the active site of calpain
Authors: Moldoveanu, T. / Hosfield, C.M. / Lim, D. / Elce, J.S. / Jia, Z. / Davies, P.L.
#2: Journal: Nat.Struct.Mol.Biol. / Year: 2003
Title: Calpain silencing by a reversible intrinsic mechanism
Authors: Moldoveanu, T. / Hosfield, C.M. / Lim, D. / Jia, Z. / Davies, P.L.
#3: Journal: Embo J. / Year: 1999
Title: Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation
Authors: Todd, B. / Moore, D. / Deivanayagam, C.C. / Lin, G.D. / Chattopadhyay, D. / Maki, M. / Wang, K.K. / Narayana, S.V.
#4: Journal: Proc.Natl.Acad.Sci.USA / Year: 2000
Title: The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium
Authors: Hosfield, C.M. / Elce, J.S. / Davies, P.L. / Jia, Z.
#5: Journal: J.Mol.Biol. / Year: 2003
Title: A structural model for the inhibition of calpain by calpastatin: crystal structures of the native domain VI of calpain and its complexes with calpastatin peptide and a small molecule inhibitor
Authors: Strobl, S. / Fernandez-Catalan, C. / Braun, M. / Huber, R. / Masumoto, H. / Nakagawa, K. / Irie, A. / Sorimachi, H. / Bourenkow, G. / Bartunik, H. / Suzuki, K. / Bode, W.
#6: Journal: J.Mol.Biol. / Year: 2004
Title: Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site
Authors: Moldoveanu, T. / Campbell, R.L. / Cuerrier, D. / Davies, P.L.
History
DepositionJun 11, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 11, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Advisory / Version format compliance
Revision 1.2Oct 25, 2017Group: Refinement description / Category: software
Revision 1.3Jan 24, 2018Group: Database references / Category: citation_author / Item: _citation_author.name
Revision 1.4Oct 20, 2021Group: Database references / Derived calculations / Category: database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Calpain-2 catalytic subunit
B: Calpain small subunit 1
C: Calpastatin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)112,56313
Polymers112,1623
Non-polymers40110
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10740 Å2
ΔGint-155 kcal/mol
Surface area41750 Å2
MethodPISA
Unit cell
Length a, b, c (Å)147.390, 147.390, 47.222
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number77
Space group name H-MP42

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Components

#1: Protein Calpain-2 catalytic subunit / Calpain-2 large subunit / Calcium-activated neutral proteinase 2 / CANP 2 / Calpain M-type / M- ...Calpain-2 large subunit / Calcium-activated neutral proteinase 2 / CANP 2 / Calpain M-type / M-calpain / Millimolar-calpain


Mass: 81577.836 Da / Num. of mol.: 1 / Mutation: C105S
Source method: isolated from a genetically manipulated source
Details: C-terminal His6 tagged / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Capn2 / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: Q07009, calpain-2
#2: Protein Calpain small subunit 1 / CSS1 / Calcium-dependent protease small subunit 1 / Calcium-dependent protease small subunit / CDPS ...CSS1 / Calcium-dependent protease small subunit 1 / Calcium-dependent protease small subunit / CDPS / Calpain regulatory subunit / Calcium-activated neutral proteinase small subunit / CANP small subunit


Mass: 21304.979 Da / Num. of mol.: 1 / Fragment: UNP residues 87-270
Source method: isolated from a genetically manipulated source
Details: tagless / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Capns1, Capn4, Css1 / Plasmid: pACpET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: Q64537
#3: Protein Calpastatin / Calpain inhibitor


Mass: 9279.284 Da / Num. of mol.: 1 / Fragment: Inhibitory domain 1, UNP residues 193-278
Source method: isolated from a genetically manipulated source
Details: tagless / Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Cast / Plasmid: pET24d / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 DE3 / References: UniProt: P27321
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 8

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Sample preparation

CrystalDensity Matthews: 2.29 Å3/Da / Density % sol: 46.21 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 5.5
Details: 4-9% PEG 3350, 5-10 mM CaCl2, 50-100 mM NaOAc (pH 5.5) , VAPOR DIFFUSION, HANGING DROP, temperature 298.0K

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Data collection

Diffraction
IDMean temperature (K)Crystal-ID
11001
21001
31001
41001
1,2,3,41
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONALS 5.0.310.98064
SYNCHROTRONNSLS X29A20.98064
SYNCHROTRONAPS 22-ID30.98064
SYNCHROTRONAPS 22-BM40.98064
Detector
TypeIDDetectorDate
ADSC QUANTUM 3151CCDJul 2, 2007
ADSC QUANTUM 3152CCD
MARMOSAIC 225 mm CCD3CCD
MARMOSAIC 300 mm CCD4CCD
Radiation
IDProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1SINGLE WAVELENGTHMx-ray1
2SINGLE WAVELENGTHMx-ray1
3SINGLE WAVELENGTHMx-ray1
4SINGLE WAVELENGTHMx-ray1
Radiation wavelengthWavelength: 0.98064 Å / Relative weight: 1
ReflectionResolution: 2.95→49.25 Å / Num. obs: 20755 / Observed criterion σ(I): 2 / Redundancy: 4.4 % / Biso Wilson estimate: 92 Å2 / Rmerge(I) obs: 0.096 / Net I/σ(I): 21.4
Reflection shellResolution: 2.95→3.06 Å / Redundancy: 4.2 % / Rmerge(I) obs: 0.82 / Mean I/σ(I) obs: 2.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
REFMAC5.2.0019refinement
PDB_EXTRACT3.005data extraction
CBASSdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 1MDW, 1DF0 domain III and DIV and 1DVI

1mdw

1df0

1dvi


Resolution: 2.95→49.15 Å / Cor.coef. Fo:Fc: 0.939 / Cor.coef. Fo:Fc free: 0.909 / SU B: 49.607 / SU ML: 0.475 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.511 / Stereochemistry target values: MAXIMUM LIKELIHOOD
RfactorNum. reflection% reflectionSelection details
Rfree0.299 1164 5.1 %RANDOM
Rwork0.229 ---
obs0.232 20755 99.83 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 94.719 Å2
Baniso -1Baniso -2Baniso -3
1--0.62 Å20 Å20 Å2
2---0.62 Å20 Å2
3---1.25 Å2
Refinement stepCycle: LAST / Resolution: 2.95→49.15 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7290 0 10 0 7300
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0227445
X-RAY DIFFRACTIONr_bond_other_d0.0020.0211
X-RAY DIFFRACTIONr_angle_refined_deg1.4851.95510055
X-RAY DIFFRACTIONr_angle_other_deg16.358318
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.1875900
X-RAY DIFFRACTIONr_dihedral_angle_2_deg39.97724.433379
X-RAY DIFFRACTIONr_dihedral_angle_3_deg22.028151314
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.9861550
X-RAY DIFFRACTIONr_chiral_restr0.1080.21080
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.025720
X-RAY DIFFRACTIONr_gen_planes_other0.0020.027
X-RAY DIFFRACTIONr_nbd_refined0.2520.23474
X-RAY DIFFRACTIONr_nbd_other0.3130.212
X-RAY DIFFRACTIONr_nbtor_refined0.3180.25043
X-RAY DIFFRACTIONr_nbtor_other0.320.219
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1790.2258
X-RAY DIFFRACTIONr_metal_ion_refined0.1720.219
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1590.228
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.3450.21
X-RAY DIFFRACTIONr_mcbond_it0.531.54593
X-RAY DIFFRACTIONr_mcbond_other0.0881.58
X-RAY DIFFRACTIONr_mcangle_it0.90427223
X-RAY DIFFRACTIONr_scbond_it1.20833245
X-RAY DIFFRACTIONr_scangle_it1.9754.52832
LS refinement shellResolution: 2.95→2.975 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.448 73 -
Rwork0.284 1580 -
all-1653 -
obs--98.69 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
15.8461-0.24340.58423.77040.24243.21060.04790.0898-0.8194-0.3152-0.26321.46580.6288-0.58440.2153-0.20670.0087-0.1201-0.2269-0.16740.51743.087-45.79314.6625
25.316-0.4795-1.36975.79881.08244.3495-0.0303-0.67591.5840.2775-0.23551.2516-0.3852-0.41670.2658-0.39530.1176-0.1022-0.1261-0.35411.01861.2813-19.722512.8288
35.37950.49080.56246.5279-1.15864.6022-0.0836-0.46880.30850.1505-0.0440.1268-0.21860.12640.1275-0.4490.08710.0235-0.4013-0.0473-0.523833.5999-33.791416.2472
43.3486-0.38240.60193.66831.14127.33620.1059-0.0934-0.41910.24670.1866-0.32670.45350.7125-0.2925-0.27960.00850.016-0.27780.0002-0.434948.3212-60.10993.6265
51.6470.47891.38276.1746-1.09032.5830.0982-0.0144-0.29850.07780.00550.88190.0159-0.1137-0.1036-0.19880.0506-0.0046-0.2567-0.0602-0.053627.9048-73.8638-0.4042
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
1X-RAY DIFFRACTION1AA24 - 21024 - 210
2X-RAY DIFFRACTION1CC170 - 19035 - 55
3X-RAY DIFFRACTION2AA211 - 340211 - 340
4X-RAY DIFFRACTION3AA360 - 500360 - 500
5X-RAY DIFFRACTION3CC162 - 16927 - 34
6X-RAY DIFFRACTION4AA540 - 701540 - 701
7X-RAY DIFFRACTION4CC137 - 1482 - 13
8X-RAY DIFFRACTION5BB97 - 27011 - 184
9X-RAY DIFFRACTION5CC211 - 22176 - 86

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