Human ubiquitin conjugating enzyme E2

Target ID:

ubc81

Aliases:

ATTPFLJ11068UEV3

Deposition Date:

Tuesday 29th August 2006

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

2I6T

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,P.J.FINERTY JR.,C.BUTLER-COLE,W.TEMPEL,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON

Site:

Toronto

ICM Datapack:

ICM Datapack

2I6T

tabs

Structure Details

During vigorous exercise or under anaerobic conditions, ATP is generated mostly by the faster glycolytic pathway, not by the citric acid cycle. But because glycolysis also produces the reduced cofactor, NADH, which would normally be oxidized by the citric acid cycle, an additional mechanism exists to very quickly regenerate NAD from NADH: reduction of pyruvate to lactate by lactate dehydrogenase (LDH). During prolonged exercise or sustained anaerobic conditions, when lactate accumulation can inhibit glycolysis, gluconeogenesis in the liver kicks-in, converting lactate to glucose. Lactate dehydrogenase is a tetramer composed of combinations of two gene products: A4, A3B1, A2B2, A1B3, and B4. Affinity for substrate and allosteric regulation differ between the two types. For example, B4 preferably converts lactate to pyruvate and is found mostly in heart muscle, where the citric acid cycle is very active, while A4 found in skeletal muscle prefers reduction of pyruvate. Four other lactate dehydrogenase gene products include LDHC, LDHAL6A, LDHAL6B, and UEV3, whose functions or biological roles are unknown.

The high resolution structure of the unliganded LDH domain of UEV3 reveals the conserved Rossman fold, with a putative active site that most closely resembles that of the Plasmodium falciparum LDH structure (Brown et al., 2004). Catalytically active forms of LDH use a conserved histidine as general acid or base during the reaction, providing a proton to the carbonyl oxygen of the substrate or acquiring a proton in the oxidative direction (Clarke et al., 1988). This residue in UEV3 is substituted by a glutamine, a residue that is not commonly used as general acid or base. Whether or not the LDH domain of UEV3 is catalytically active is not yet known (Kloor et al., 2002).

Materials and Methods

UEV3 LDH domain

PDB:2I6T

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Open Biosystems
SGC Clone Accession: ubc81.171.471; plate SDC092:H7
Tag:N-terminal His-tag with integrated TEV-cleavage site: MHHHHHHSSGRENLYFQG.
Host:E.coli BL21 (DE3)

Construct

Prelude:
Sequence:MHHHHHHSSGRENLYFQGSKSWANHENKTVNKITVVGGGELGIACTLAISAKGIADRLVLLDLSEGTKGATMDLEIFNLPNVEISKDLSASAHSKVVIFTVNSLGSSQSYLDVVQSNVDMFRALVPALGHYSQHSVLLVASQPVEIMTYVTWKLSTFPANRVIGIGCNLDSQRLQYIITNVLKAQTSGKEVWVIGEQGEDKVLTWSGQEEVVSHTSQVLSNRAMELLRVKGQRSWSVGLSVADMVDSIVNNKKKVHSVSALAKGYYDINSEVFLSLPCILGTNGVSEVIKTTLKEDTVTEKLQSSASSIHSLQQQLKL
Vector:p28a-mhl

Growth

Medium:
Antibiotics:
Procedure:UEV3 LDH domain was expressed in E. coli BL21 (DE3) grown in Terrific Broth (Sigma) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 5.5-6. Protein expression was then induced by isopropyl-1-thio-D-galactopyranoside at a final concentration of 0.05 mM, and incubation continued overnight at 15ºC. The culture was centrifuged; the cell pellets were collected, frozen with liquid nitrogen and stored at -80ºC.

Purification

Procedure
Column 1: TALON metal-affinity resin column (BD Biosciences) .
Column 2: HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham)

The cleared lysate was loaded onto a TALON metal-affinity resin (BD Biosciences) column at 4ºC. The column was washed with wash buffer A, wash buffer B and again wash buffer A, and the protein was eluted with elution buffer. The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with 20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5% glycerol, 2 mM dithiothreitol and concentrated by ultrafiltration.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer and lysed using Microfluidizer (peak pressure 18,000 psi). The lysate was cleared by centrifugation.
Concentration:
Ligand
MassSpec:
Crystallization:Purified UEV LDH domain was crystallized using the hanging-drop vapor-diffusion method. Crystals grew when the protein (6 mg/ml in the above gel-filtration buffer) was mixed with the reservoir solution in a 1:1 volume ratio, and the drop was equilibrated against a reservoir solution containing 26% PEG3350, 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 5.2 at 293K temperature.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Brown,W.M., Yowell,C.A., Hoard,A., Jagt,T.A.V., Hunsaker,L.A., Deck,L.M., Royer,R.E., Piper,R.C., Dame,J.B., Makler,M.T., and Jagt,D.L.V. (2004). Comparative structural analysis and kinetic properties of lactate dehydrogenases from the four species of human malarial parasites. Biochemistry 43, 6219-6229.
Clarke,A.R., Wilks,H.M., Barstow,D.A., Atkinson,T., Chia,W.N., and Holbrook,J.J. (1988). An Investigation of the Contribution Made by the Carboxylate Group of An Active-Site Histidine Aspartate Couple to Binding and Catalysis in Lactate-Dehydrogenase. Biochemistry 27, 1617-1622.
Kloor,M., Bork,P., Duwe,A., Klaes,R., Doeberitz,M.V., and Ridder,R. (2002). Identification and characterization of UEV3, a human cDNA with similarities to inactive E2 ubiquitin-conjugating enzymes. Biochimica et Biophysica Acta-Gene Structure and Expression 1579, 219-224.