Human calpain 9

Target ID:

CAPN09

Aliases:

GC36nCL-4

Deposition Date:

Thursday 1st March 2007

Families:

Proteases

Related Diseases:

SignallingCancer

Structure type:

protein-ligand

Download Coordinates:

2P0R

Authors:

T.L.DAVIS,R.PARAMANATHAN,J.R.WALKER,C.BUTLER-COLE,P.J.FINERTY JR.,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Toronto

2P0R

tabs

Structure Details

Calpains are intracellular cysteine proteases involved in signal transduction. Pathways that increase calcium levels lead to calpain activation.
There are multiple mechanisms by which calpains are kept suppressed in the absence of calcium.
Consequently, there are many calcium-induced activation mechanisms; these have been the focus of numerous recent studies.
Notably, the papain-like catalytic domain is composed of two lobes, each containing a structural and regulatory calcium binding site [1]. The relative orientation between lobes as well as the conformation of a loop in the amino-terminal lobe (adjacent to the active site) are partially controlled by carboxy-terminal domains such as penta-EF hands and C2-like domains [2,3]. Full catalytic activity therefore requires regions in addition to the canonical papain-like domain.

Calpain-9 disregulation has been implicated in stomach cancer and hypertension [4-6]. Detailed studies of this protein may therefore benefit related therapeutic programs. We have recently determined the structure of the canonical catalytic core of calpain 9 and found an interlobe orientation that suggest an additional component to the mechanism of calpain 9 activation [7]. Here we describe the structure of the leupeptin-bound complex of calpain 9, providing a snapshot of a potential intermediate along the reaction mechanism of this enzyme.

Materials and Methods

CAPN9 + leupeptin

PDB:2P0R

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:AAB80762
Entry Clone Source:capn09.NM_006615.GSC.capn09.001.365.vec
SGC Clone Accession:capn09.010.340.02D05
Tag:N-terminal histag: mgsshhhhhhssglvprgs
Host:BL21 (DE3)

Construct

Prelude:
Sequence:gsPQAHPVPKDARITHSSGQSFEQMRQECLQRGTLFEDADFPASNSSLFYSERPQIPFVWKRPGEIVKNPEFILGGATRTDICQGELGDCWLLAAIASLTLNQKALARVIPQDQSFGPGYAGIFHFQFWQHSEWLDVVIDDRLPTFRDRLVFLHSADHNEFWSALLEKAYAKLNGSYEALKGGSAIEAMEDFTGGVAETFQTKEAPENFYEILEKALKRGSLLGCFIDTRSAAESEARTPFGLIKGHAYSVTGIDQVSFRGQRIELIRIRNPWGQVEWNGSWSDSSPEWRSVGPAEQKRLCHTALDDGEFWMAFKDFKAHFDKVEICNLTPDA
Vector:p28a-thrombin-LIC

Growth

Medium:TB
Antibiotics:
Procedure:Overnight cultures were grown from glycerol stocks in LB media. Large-scale inolculations were performed by adding 50 mLs of overnight culture into 900 mL of TB media supplemented with 1.5% glycerol, 50 µg/ ml kanamycin and 300 µL antifoam 204 (Sigma A-8311). Cells were grown at 37 °C using the LEX bubbling system until OD reaches ~5, at which time the temperature of the cultures were reduced to 15 °C. Cultures were induced when the cell density was between 6-8 with a final concentration of 100 µM IPTG and left overnight. Cells were harvested by centrifugation at 12,000xg for 15 minutes.

Purification

Procedure
Lysed cells were diluted 1:1 in lysis buffer; imidazole was added to a final concentration of 30 mM, and 1.5 mL of his-Link resin (Promega) was added to the lysate. This mixture was then rocked at 4 degC for 1 hour. After incubation for 1 hour, the mixture was centrifuged at 500xg and the supernatant carefully decanted. The resin was washed four times with IMAC buffer, then resuspended and loaded into a gravity column. The resin was washed with 5 column volumes of low imidazole buffer and then eluted with 10 mLs of elution buffer. Two units of thrombin (Sigma) per milligram of protein were added and incubated overnight at 4 degC to cleave the (His)6 tag. The cut protein was then loaded onto an XK 16x65 column packed with highLoad Superdex 200 resin (GE Healthcare). Peak fractions were pooled and concentrated using Amicon concentrators with 10,000 MWCO (Millipore) to 20 mg/mL.

Extraction

Procedure
Frozen cell pellets were thawed on ice. Each pellet was resuspended in 25 mL lysis buffer, homogenized, and lysed using sonication.
Concentration:
Ligand
MassSpec:
Crystallization:Purified protein at a concentration of 10 mg/mL was incubated with 10-fold molar excess of leupeptin (Sigma) overnight. Crystals were obtained in hanging drop plates at 18 degC using the follow crystallization conditions: 14.7% PEG8000, 10% glycerol, 0.1M sodium cacodylate pH 5.5, and 0.15M (NH₄)₂SO₄. Crystals were harvested into cryoprotectant containing 15% glycerol and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Moldoveanu,T., Jia,Z. & Davies,P.L. Calpain activation by cooperative Ca2+ binding at two non-EF-hand sites. J. Biol. Chem. 279, 6106-6114 (2004).
Moldoveanu,T. et al. A Ca(2+) switch aligns the active site of calpain. Cell 108, 649-660 (2002).
Moldoveanu,T., Hosfield,C.M., Lim,D., Jia,Z. & Davies,P.L. Calpain silencing by a reversible intrinsic mechanism. Nat. Struct. Biol. 10, 371-378 (2003).
Yoshikawa,Y., Mukai,H., Hino,F., Asada,K. & Kato,I. Isolation of two novel genes, down-regulated in gastric cancer. Jpn. J. Cancer Res. 91, 459-463 (2000).
Markmann,A. et al. Down-regulation of calpain 9 is linked to hypertensive heart and kidney disease. Cell Physiol Biochem. 15, 109-116 (2005).
Liu,K., Li,L. & Cohen,S.N. Antisense RNA-mediated deficiency of the calpain protease, nCL-4, in NIH3T3 cells is associated with neoplastic transformation and tumorigenesis. J. Biol. Chem. 275, 31093-31098 (2000).
Davis,T.L. et al. The Crystal Structures of Human Calpains 1 and 9 Imply Diverse Mechanisms of Action and Auto-inhibition. J. Mol. Biol. (2006).