Human calpain 7 C2 domain

Target ID:

capn08

Aliases:

CALPAIN7FLJ36423PALBH

Deposition Date:

Wednesday 27th June 2007

Families:

Proteases

Related Diseases:

Neurobiology

Structure type:

apo

Download Coordinates:

2QFE

Authors:

Walker JR, Cuerrier D, Paramanathan R, Weigelt J, Arrowsmith CH , Edwards AM, Bochkarev A, Dhe-Paganon S.

Site:

Toronto

ICM Datapack:

ICM Datapack

2QFE

tabs

Structure Details

Cuerrier, D., Walker, J.R., Paramanathan, R., Weigelt, J., Arrowsmith, C.H., Edwards, A.M., Bochkarev, A., Dhe-Paganon, S.

Calpains are a family of calcium-dependent intracellular proteases that participate in signal transduction pathways by cleaving target proteins when local calcium concentrations rise beyond a certain threshold. Reported calpain substrates range from cytoskeletal proteins (alpha-spectrin) to membrane pumps (the Na+/Ca2+ exchanger NCX), apoptosis-related proteins (caspase-7 and -9) to transcription factors (c-Jun, c-Fos) and cell-cycling proteins (p35) [1]. The proteolytic activity of calpains is tightly controlled within the cell by several levels of regulation involving multiple non-catalytic domains. In addition to being regulated by calcium levels, calpain activity is regulated by autoproteolysis, membrane localization, phosphorylation, interactions with scaffolding proteins, and interactions with its endogenous inhibitor, calpastatin [1].

All but one of the 13 human calpain isoforms contain a characteristic domain carboxy-terminal to the proteolytic core called the calpain C2-like domain. The name reflects its structure first observed in calpain 2 as an 8-stranded beta-sandwich bearing superficial similarity to the C2 domain of protein kinase C and synaptotagmin [2;3]. The C2-like domains of calpains 1 and 2 provide a structural scaffold that is important for the calcium-induced activation of the proteolytic domain via multiple electrostatic interactions with the acidic and basic loops of the C2-like domain [4]. Although canonical C2 domains can combine calcium-binding and phospholipid-binding activities, there is contradictory evidence of such a role for the C2-like domain of calpains.

Calpain 7 is a ubiquitous isoform whose physiological role remains unknown, although its activated form has been observed in the nucleus of a cell model of Huntingtonâ€™s disease [5]. Whereas the majority of calpains contain a regulatory penta-EF-hand (PEF) domain at their C-terminus, a small subset of calpains, including calpain 7 [6], lack PEF domains but instead have a second, tandem C2-like domain. Spacial restrictions indicate that this distal C2-like domain has a role separate from the structural support for the proteolytic core of the proximal C2-like domain, providing a novel layer to the regulation of calpain activity.

We here present the structure of the distal C2-like domain of calpain 7. The structure shows high degree of structural divergence from previous calpain C2-like domains, especially in the loop connecting beta4 and beta5 strands. Notably, this loop lacks helix alpha2 and forms a novel beta-strand with beta4. The absence of the acidic and basic loops that forms extensive contacts with the proteolytic core in other structures suggests that the distal C2-like domain performs a different role than the latter. Moreover, the absence of coordinated calcium in the structure despite its presence in the crystallization buffer strongly suggests that this domain does not directly bind calcium, contrary to previous models of calpain C2-like domains. This structure serves to further emphasize the structural divergence of calpain C2-like domains from canonical C2 domains, and provides insight into the role of this domain in the regulation of calpain activity.

Materials and Methods

CAPN7

PDB:2QFE

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_055111
Entry Clone Source:capn08.BC056202.MGC.AT63C1.pBluescript
SGC Clone Accession:capn08.685.813; plate SDC059F06
Tag:MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPRGSPYTLSKRINGKWSGQSAGGCGNFQETHKNNPIYQFHIEKTGPLLIELRGPRQYSVGFEVVTVSTLGDPGPHGFLRKSSGDYRCGFCYLELENIPSGIFNIIPSTFLPKQEGPFFLDFNSIIPIKITQLQ
Vector:pET28a-LIC

Growth

Medium:
Antibiotics:
Procedure:A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37oC. Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37oC. The temperature of the media was reduced to 15oC one hour prior to induction and induced at OD(600) = 6 with 100 uM isopropyl-thio--D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15oC, and cell pellets collected by centrifugation and frozen at -80 oC.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of HisLink Protein Purification Resin (Promega V8821) per 40 mL lysate. The mixture was incubated with mixing for at least 20 minutes at 4oC. The lysate was spun at 500 xg for 5 minutes, and the supernatant was decanted. The remaining resin was washed with 45 mL of cold wash buffer, allowing 5 minutes to settle between washes, until the supernatant was clear (usually 3-5 washes). The washed resin was transferred to a gravity column and further washed with 1 column volumes (approx. 5 mL) of wash buffer at approximately 3 mL/min. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10 mL) of elution buffer. Gel filtration 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) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 3 mL/min. The eluate sample from the IMAC step (approx. 10 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.Protein concentration. Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration of 10-15 mg/mL.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals of the calpain 7 distal C2-like domain were grown at 298 K using the hanging drop method by mixing equal volumes of 1.4 M NaKPO4 pH 7.0 and 13 mg/mL protein. The crystals were cryoprotected by dragging through a 50% mixture of mineral oil and paratone.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

[1] D.E. Goll, V.F. Thompson, H. Li, W. Wei, J. Cong, The calpain system. Physiol Rev. 83 (2003) 731-801.

[2] C.M. Hosfield, J.S. Elce, P.L. Davies, Z. Jia, Crystal structure of calpain reveals the structural basis for Ca(2+)-dependent protease activity and a novel mode of enzyme activation. EMBO J. 18 (1999) 6880-6889.

[3] S. Strobl, C. Fernandez-Catalan, M. Braun, R. Huber, H. Masumoto, K. Nakagawa, A. Irie, H. Sorimachi, G. Bourenkow, H. Bartunik, K. Suzuki, W. Bode, The crystal structure of calcium-free human m-calpain suggests an electrostatic switch mechanism for activation by calcium. Proc.Natl.Acad.Sci.U.S.A. 97 (2000) 588-592.

[4] C.M. Hosfield, T. Moldoveanu, P.L. Davies, J.S. Elce, Z. Jia, Calpain mutants with increased Ca2+ sensitivity and implications for the role of the C(2)-like domain. J.Biol.Chem. 276 (2001) 7404-7407.

[5] J. Gafni, E. Hermel, J.E. Young, C.L. Wellington, M.R. Hayden, L.M. Ellerby, Inhibition of calpain cleavage of huntingtin reduces toxicity: accumulation of calpain/caspase fragments in the nucleus. J.Biol.Chem. 279 (2004) 20211-20220.

[6] T. Franz, M. Vingron, T. Boehm, T.N. Dear, Capn7: a highly divergent vertebrate calpain with a novel C-terminal domain. Mamm.Genome 10 (1999) 318-321.