Human calpain 8

Target ID:

CAPN08a

Aliases:

nCL-2

Deposition Date:

Monday 30th October 2006

Families:

Proteases

Related Diseases:

SignallingCancer

Structure type:

apo

Download Coordinates:

2NQA

Authors:

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

Site:

Toronto

ICM Datapack:

ICM Datapack

2NQA

tabs

Structure Details

Calpains are calcium-activated, intracellular cysteine proteases. Some are implicated in signal transduction, amplifying or quenching signals by cleaving key intermediates.
For example, proteolysis in immune cells of IKB by calpain-1, which is in turn activated by upstream calcium signaling, leads to activation of inflammatory pathways and in some cases antiapoptosis.
On the other hand, injury-induced calcium influxes in neuronal tissue leads to calpain activiation, destruction of cytoskeletal, nuclear and cytosolic proteins, and subsequent cell death.
Moreover, mutations in calpain 3 cause limb-girdle muscular dystrophies 1; wild type, active calpain 3 is necessary for proper skeletal muscle maintenance.

The exact molecular functions, including substrate specificity and subcellular localization of calpains are poorly understood. Recent studies have shown that calpains are regulated by many different mechanisms, including calcium levels, autoproleolysis, membrane localization, and phosphorylation. Calcium activates through a complex series of events that, for many calpains, is initiated by binding to a common regulatory subunit and the regulatory domain at the Carboxy-terminus of the protein, both formed by a similar penta-EF-hand fold 2. This leads to release of constrictions to the catalytic domain and allosteric-binding of additional calcium atoms near the active site. Dysfunction of these controls are link to numerous diseases. Structures of calpains will enlighten related mechanisms and potential avenues of therapy.

Calpain 8 is predominantly expressed in the stomach [3], specifically in mucus cells in the gastric epithelium and duodenum [4] and the pituitary [5]. The biological role or signaling function of calpain 8 is far from understood. Preliminary evidence points to a regulatory role in the secretory process, whether secretion of neutralizing mucus from pit cells 4 or gonadotropins from pituitary cells 5. Yeast-2-hybrid experiments have identified the β-coatomer as a potential scaffold and substrate for calpain 8 [4].

We present here the high resolution structure of the catalytic domain of calpain 8 (aka, nCL-2) in complex with leupeptin.
The two lobes (IIa/IIb) are oriented with respect to each other in an active conformation,
with the two calciums bound at their canonical sites, Arg94-Glu323 forming inter-lobe salt bridge, and the catalytic triad propped in an active conformation.
A hemithioacetal bond is formed between the catalytic cysteine and leupeptin.
Unlike the calpain-1-leupeptin structure (PDB 1TL90), the arginine side chain of leupeptin does not interact with and close the amino-terminal gating loop 6; instead it forms a salt bridge with Glu257 in the C-terminal gating loop. The leucyl side chain of leupeptin is stabilized within the S2 pocket by van der Walls interactions with Trp106, Ala263, Ser241, and Ser199.
Backbone amides of leupeptin form hydrogen bonds with Gly197 and Ser261. Further characterization of this complex structure may offer an additional template for structure-based drug design.

Materials and Methods

Human calpain 8

PDB:2NQA

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:
SGC Clone Accession:capn08a.023.346; plate SDC072 D11
Tag:Thrombin cleavable N-term his tag
Host:

Construct

Prelude:
Sequence:mgsshhhhhhssglvprgsNALKYLGQDFKTLRQQCLDSGVLFKDPEFPACPSALGYKDLGPGSPQTQGIIWKRPTELCPSPQFIVGGATRTDICQGGLGDCWLLAAIASLTLNEELLYRVVPRDQDFQENYAGIFHFQFWQYGEWVEVVIDDRLPTKNGQLLFLHSEQGNEFWSALLEKAYAKLNGCYEALAGGSTVEGFEDFTGGISEFYDLKKPPANLYQIIRKALCAGSLLGCSIDVYSAAEAEAITSQKLVKSHAYSVTGVEEVNFQGHPEKLIRLRNPWGEVEWSGAWSDDAPEWNHIDPRRKEELDKKVEDGEFWMSLSDFVRQFSRLEICNLSPDSLS
Vector:

Growth

Medium:
Antibiotics:
Procedure:Inoculation: The appropriate well of a 96 well block from the -80oC freezer containing a glycerol stock of the bacteria of interest is pierced and plunged with a needle by a flame. The needle is dropped into a 250 ml flask containing 100 mL of LB (Sigma L7658) supplemented with 50 mg/ ml kanamycin (BioShop Canada KAN 201). The flask is shaken overnight (16 hours) at 250 rpm at 37oC. A 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918), supplemented with 1.5% glycerol, 50 mg/ ml kanamycin and 300 ul antifoam 204 (Sigma A-8311 is inoculated by a flame by pouring in the overnight LB culture. A sterilized cap/sparger (Fisher 11-138B) assembly is then inserted into the bottle.

Induction: The temperature of the media begins to be reduced to 15oC one hour prior to induction so that the media is at 15oC at the time of induction. Cultures are induced when the OD(600) reaches between 4 and 8 with 100 mM isopropyl-thio-b-D-galactopyranoside (BioShop Canada IPT 001), in the absence of flame. Cultures continue to be aerated overnight (16 hours) at 15oC. Aeration is slightly reduced at this time to avoid over foaming of the lower temperature culture.

Purification

Procedure

IMAC purification: The lysate is spun at 500xg for 3 minutes to pellet the HisLink resin. The lysate is carefully decanted off the resin, and then 50 mL of lysis buffer are added to wash the resin. The resin is allowed to settle for 5 minutes, then poured off and washed 3 more times with fresh lysis buffer. The washed resin is then loaded onto a gravity column, and then washed with a column volume of low imidazole buffer (lysis buffer + 30 mM imidazole pH 8). A 4 mL sample of the low imidazole wash is saved for later analysis by SDS-PAGE. Samples are eluted from the HisLink resin by exposure to 10 mL elution buffer (lysis buffer + 500 mM imidazole and 10% glycerol) at a 1mL/min flow rate. A 10 uL sample of the eluate is saved for SDS-PAGE analysis. 10 uL of each eluate is saved for measurement of protein concentration using Bradford.

(His)6-tag cleavage: 1 unit of thrombin protease per milligram of protein is added to the sample. The conical vial is stored without shaking, overnight, at 4oC.

Size exclusion chromatography: An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) is pre-equilibrated with gel filtration buffer (lysis buffer + 5 mM b-ME and 1mM EDTA) for 1.5 column volumes using an AKTAxpress at a flow rate of 3 mL/min. 10 mL of sample is loaded onto the column at 1.5 mL/min, and 2mL fractions are collected into 96-well plates using peak fractionation protocols. Peak fractions are analyzed for purity using SDS-PAGE and/or mass spectrometry and pooled.

Protein concentration: Purified proteins are concentrated using either 4 mL or 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, UFC901024 or 5,000 MWCO, UFC900524, as appropriate, Millipore) to a final concentration of 20 mg/mL for crystallographic screening or other biophysical studies.

Extraction

Procedure
Frozen cell pellets contained in bags (Beckman 369256) obtained from 2L liters of culture are thawed by soaking in warm water. Each cell pellet is resuspended in 20 mL lysis buffer (50 mM Tris pH 8.0 and 500 mM NaCl 1mM phenylmethanesulfonyl fluoride and 1mL Sigma general protease inhibitor (Sigma P2714, resuspended according to manufacturerÂs instructions) and then homogenized at maximal setting for 30-60 seconds per pellet. Cell lysis is accomplished by sonication on ice: the sonication protocol is 10 sec pulse at half-maximal frequency, 10 second rest, for 6 minutes total sonication time per pellet. Lysed cells are diluted to 50 -100 mL final volume and imidazole added to a final concentration of 10 mM; this is then mixed with 2-3 mL of HisLink Protein Purification Resin (Promega V8821) per construct. The mixture is incubated with mixing for at least 20 minutes at 4oC.
Concentration:
Ligand
MassSpec:
Crystallization:Protein concentration: 10 mg/mL
Crystallization method: Sitting drop vapor diffusion
Crystallization conditions: 2M NH4PO4, 0.1M Tris, pH 8.5
Crystallization temperature: 18 degC
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Zatz,M. & Starling,A. Mechanisms of disease: Calpains and disease. N. Engl. J. Med. 352, 2413-2423 (2005).
Suzuki,K., Hata,S., Kawabata,Y. & Sorimachi,H. Structure, activation, and biology of calpain. Diabetes 53, S12-S18 (2004).
Sorimachi,H., Ishiura,S. & Suzuki,K. A novel tissue-specific calpain species expressed predominantly in the stomach comprises two alternative splicing products with and without Ca(2+)-binding domain. J. Biol. Chem. 268, 19476-19482 (1993).
Hata,S. et al. 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 (2006).
Duan,W.R., Ito,M., Lee,E.J., Chien,P.Y. & Jameson,J.L. Estrogen regulates a tissue-specific calpain in the anterior pituitary. Biochem. Biophys. Res. Commun. 295, 261-266 (2002).
Moldoveanu,T., Campbell,R.L., Cuerrier,D. & Davies,P.L. Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site. J. Mol. Biol. 343, 1313-1326 (2004).