Human ubiquitin-conjugating enzyme E2-like, LDH domain

Target ID:

ubc81

Aliases:

ATTPFLJ11068UEV3

Deposition Date:

Thursday 26th June 2008

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

3DL2

Authors:

Walker JR, Avvakumov G, Xue S, Newman EM, Finerty PJ, Butler-Cole C, Bountra C, Wikstrom M, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S.

Site:

Toronto

3DL2

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

PDB:3DL2

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:ubc81.LIFESEQ2844953.OBS.13511-G-4.pINCY
Entry Clone Source:Open Biosystems
SGC Clone Accession:ubc81.171.471; plate SDC076H08
Tag:MGSSHHHHHHSSGLVPR*GS
Host:E. coli BL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPR*GSSKSWANHENKTVNKITVVGGGELGIACTLAISAKGIADRLVLLDLSEGTKGATMDLEIFNLPNVEISKDLSASAHSKVVIFTVNSLGSSQSYLDVVQSNVDMFRALVPALGHYSQHSVLLVASQPVEIMTYVTWKLSTFPANRVIGIGCNLDSQRLQYIITNVLKAQTSGKEVWVIGEQGEDKVLTWSGQEEVVSHTSQVQLSNRAMELLRVKGQRSWSVGLSVADMVDSIVNNKKKVHSVSALAKGYYDINSEVFLSLPCILGTNGVSEVIKTTLKEDTVTEKLQSSASSIHSLQQQLKL
Vector:PET28a-LIC

Growth

Medium:Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in growth medium at 37®C to an OD₆₀₀ of 5.5. Protein expression was induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15®C. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80®C.

Purification

Procedure
The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 10 mL Wash buffer A, 10 mL Wash buffer B, and 10 mL Wash buffer A. The protein was eluted with 6 mL Elution buffer. His-tag was removed by incubation of the protein with thrombin (1 U per mg protein, overnight at 4 °C). The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by SDS-PAGE) were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown in hanging drops by mixing 2 ul UBE2V3 solution (6 mg/ml) with 2 ul well solution (1.6 M Na/KPO4, pH 6.5, 0.2 M NaCl, 2 mM DTT) at 18°C. The crystals were cryoprotected by immersion in the well solution mixed in 1:1 ratio with a water solution containing 20% (w/v) sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethylene glycol.
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.