human autophagy-related cysteine endopeptidase 2 isoform a; Homo sapiens ATG4 autophagy related 4 homolog A

Target ID:

APG4A

Aliases:

APG4AAUTL2

Deposition Date:

Wednesday 21st March 2007

Families:

Proteases

Related Diseases:

MetabolismCancerNeurobiology

Structure type:

apo

Download Coordinates:

2P82

Authors:

J.R.WALKER,T.DAVIS,S.MUJIB,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

2P82

tabs

Structure Details

During the heyday of electron microscopy (1960's) autophagic vacuoles were commonly observed in cells. These ultrastructures were described as membranes that envelope organelles and isolation bodies formed from invested organelles. Fusion with lysosomes then result in the formation of autophagic vacuoles. The end point is degradation of the ingested organelles back into their basic building block for recycling. Today the component enzymes in this complex machinery are beginning to be identified and some insight into the mechanism of this process is being uncovered.

The process of autophagy is catalyzed by a set of proteins similar in function but unrelated in sequence to those in the ubiquitylation pathway. The key difference is the target. While the ubiquitylation pathway is directed towards individual protein targets, the autophagic pathway is directed towards large protein complexes and organelles. Ubiquitin-like modifiers are covalently attached to phospholipids in the case of organelle targets, an isopeptide bond formed between the carboxy-terminal glycine of the modifier and the amino group of phosphatidylethanolamine, for example. And reminiscent of the ubiquitylation system, when an organelle or membrane envelope is coated with enough modifiers, it is destined for fusion with lysosomes and subsequently degraded. The functional parallels continue. Ubiquitin is derived from a longer precursor, processed by maturases; ubiquitin homologs in the autophagy pathway are likewise processed by maturases. Autophagy maturases include four human paralogs: Atg4a, b, c, and d.

We have determined the high resolution structure of human Atg4a, revealing a papain-like fold (central helix and adjacent beta-sheet), catalytic triad (composed of Cys77, His281, and Asp279), and an S1 pocket (centered on Met78 but apparently blocked by the neighboring loop (Lys260-Asp263)).

Materials and Methods

Apg4a

PDB:2P82

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC061696
Entry Clone Source:Open Biosystems
SGC Clone Accession:apg4a.023.353;SDC094:C5
Tag:
Host:E.coliBL21 (DE3)

Construct

Prelude:
Sequence:

MHHHHHHSSGRENLYFQGPDTDELVWILGKQHLLKTEKSKLLSDISARLWFTYRRKFSPIGGTGPSSDAGWGCMLRCGQMMLAQALICRHLGRDWSWEKQKEQPKEYQRILQCFLDRKDCCYSIHQMAQMGVGEGKSIGEWFGPNTVAQVLKKLALFDEWNSLAVYVSMDNTVVIEDIKKMCRVLPLSADTAGDRPPDSLTASNQSKGTSAYCSAWKPLLLIVPLRLGINQINPVYVDAFKECFKMPQSLGALGGKPNNAYYFIGFLGDELIFLDPHTTQTFVDTEENGTVNDQTFHCLQSPQRMNILNLDPSVALGFFCKEEKDFDNWCSLVQKEILKENLRMFELVQKHPSHW

Vector: pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:Apg4a was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) with 50 µg/ml of kanamycin at 37ºC to an OD600 of 7.5. Cells were induced by isopropyl-1-thio-D-galactopyranoside (IPTG) at 0.1 mM overnight at 15ºC. Cell pellets were harvested by centrifugation and stored at -80ºC.

Purification

Procedure
The lysate was 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 4degC. 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 is 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. Samples are eluted from the HisLink resin by exposure to 10-14 mL elution buffer at a 1mL/min flow rate. To cut the (His)6 tag off of the protein, 5 units of TEV protease per milligram of protein is added to the sample and is stored without shaking, overnight, at 4degC. 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 for 1.5 column volumes using an AKTAxpress at a flow rate of 3 mL/min. 5 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.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer (10 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 10 mM imidazole and protease inhibitors (0.1µM phenylmethyl sulfonyl fluoride and Sigma general protease inhibitor (Sigma P2714, resuspended and diluted according to manufacturerÂs instructions) and lysed using sonication (10 sec pulse at maximal frequency, 10 second rest, for 6 minutes total sonication time, Virsonic).
Concentration:
Ligand
MassSpec:
Crystallization:Purified proteins are concentrated using 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, UFC901024) to a final concentration of 10 mg/mL for crystallography. The experimental conditions were sitting drop, 18oC, 23.5% PEG3350, 0.2M NaCl, 0.1M SPG buffer pH5.4, 5% ethylene glycol. Cryoprotection was achieved using 20% ethylene glycol.
NMR Spectroscopy:
Data Collection:
Data Processing: