Human UBCE2I-HIP2 protein-protein complex

Target ID:

UBC32

Aliases:

C358B7.1P18UBC9

Deposition Date:

Wednesday 29th November 2006

Families:

Ubiquitin Related BiologyUbiquitylation - E2 Conjugate Enzymes

Related Diseases:

Signalling

Structure type:

protein-protein complex

Download Coordinates:

2O25

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,E.M.NEWMAN,F.MACKENZIE,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

2O25

tabs

Structure Details

HIP2 (UBE2K) is an E2 involved in the production of K48 poly-ubiquitin chains and proteasome-mediated degradation. Catalytic activity does not require E3 ligases nor target substrates [1-3]. Potentially, HIP2 could be a gateway for general proteasomal degradation, a biological role which has not yet been established. HIP2 has been associated with a number of neurological processes and diseases. For example, degradation of Huntingtin may be dependant on HIP2 [4]. And HIP2 is over-expressed in Alzheimerâ€™s brains [5] and its transfection in cultured neural cells significantly increased Amyloid-β1-42 dependant apoptosis; its removal completely abolished apoptosis [6].

We have determined the first crystal structure of HIP2, showing that the UBA domain is tightly packed against the core E2 domain. To explore the possibility that HIP2 acts in concert with other E2 proteins, analogous to the UBC13/UEV2 enzyme pair, which catalyze K63 poly-ubiquitin chains, we used a simple pull-down screen against human E2 paralogs. We discovered that HIP2 interacts tightly and specifically with UBC9 (UBE2I). This interaction is consistent with recent findings showing HIP2 to be sumoylated [7], an event postulated to prevent its E1-mediated ubiquitin loading. But the physiological relevance is unknown. Our structure may shed light on this and other potential mechainsms involving ubiquitin-sumo cross-talk.

Materials and Methods

Human UBCE2I + HIP2 complex

PDB:2O25

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi21536483 (HIP2); gi42659538 (ubc9)
Entry Clone Source:MGC
SGC Clone Accession:UBE2I: ubc32.001.158.53F03
HIP2: ubc45.001.200.06B08
Tag:N-Terminal His-tag with integrated thrombin-cleavage site:
mgsshhhhhhssggvprGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:UBCE2I:
mgsshhhhhhssggvprGSMSGIALSRLAQERKAWRKDHPFGFVAVPTKNPDGTMNLMNWECAIPGKKGTPWEGGLFKLRMLFKDDYPSSPPKCKFEPPLFHPNVYPSGTVCLSILEEDKDWRPAITIKQILLGIQELLNEPNIQDPAQAEAYTIYCQNRVEYEKRVRAQAKKFAPS
HIP2:
mgsshhhhhhssggvprGSMANIAVQRIKREFKEVLKSEETSKNQIKVDLVDENFTELRGEIAGPPDTPYEGGRYQLEIKIPETYPFNPPKVRFITKIWHPNISSVTGAICLDILKDQWAAAMTLRTVLLSLQALLAAAEPDDPQDAVVANQYKQNPEMFKQTARLWAHVYAGAPVSSPEYTKKIENLCAMGFDRNAVIVALSSKSWDVETATELLLSN
Vector:p28a-LIC-thrombin

Growth

Medium:TB
Antibiotics:
Procedure:The proteins were individually expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37 degC to an OD₆₀₀ of 7.5. Protein expression was then induced with isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 0.05 mM, and incubated overnight at 15 degC. The culture was centrifuged and the cell pellets were collected and stored at -80 degC.

Purification

Procedure
UBE2I and HIP2 were purified as individual proteins as follows. The cleared lysate was loaded onto a TALON metal-affinity resin column (BD Biosciences) at 4 degC (1.5 ml settled gel volume per liter original cell culture). The column was washed with 10 ml wash buffer A, 10 mlwash buffer B and then with 30 ml wash buffer A, and the protein was eluted with 6 ml elution buffer. His-tags were removed by incubation of the proteins with thrombin, and they were combined in an equimolar ration and further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with GF buffer and concentrated by ultrafiltration to a final protein concentration of 26 mg/ml using Amicon Ultra centrifugal filter with 10kD cutoff.

Extraction

Procedure
The cell pellet was resuspended in lysis buffer containing protease inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were grown in hanging drops by mixing 2 microL protein solution with 2 microL well solution (16% PEGMME 5000, 0.1 M bis-Tris-HCl, pH 6.0, 1 mM DTT) at 21 degC. For cryoprotection, the crystals were soaked in well solution supplemented with 20% ethylene glycol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

L.D. Mastrandrea, E.M. Kasperek, E.G. Niles, C.M. Pickart, Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K. Biochemistry 37 (1998) 9784-9792.
M.T. Haldeman, G. Xia, E.M. Kasperek, C.M. Pickart, Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element. Biochemistry 36 (1997) 10526-10537.
T. Yao, R.E. Cohen, Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme. J.Biol.Chem. 275 (2000) 36862-36868.
S.J. Lee, J.Y. Choi, Y.M. Sung, H. Park, H. Rhim, S. Kang, E3 ligase activity of RING finger proteins that interact with Hip-2, a human ubiquitin-conjugating enzyme. Febs Letters 503 (2001) 61-64.
S. Song, Y.K. Jung, Alzheimer's disease meets the ubiquitin-proteasome system. Trends Mol.Med. 10 (2004) 565-570.
S. Song, S.Y. Kim, Y.M. Hong, D.G. Jo, J.Y. Lee, S.M. Shim, C.W. Chung, S.J. Seo, Y.J. Yoo, J.Y. Koh, M.C. Lee, A.J. Yates, H. Ichijo, Y.K. Jung, Essential role of E2-25K/Hip-2 in mediating amyloid-beta neurotoxicity. Molec.Cell 12 (2003) 553-563.
A. Pichler, P. Knipscheer, E. Oberhofer, W.J. van Dijk, R. Korner, J.V. Olsen, S. Jentsch, F. Melchior, T.K. Sixma, SUMO modification of the ubiquitin-conjugating enzyme E2-25K. Nature Structural & Molecular Biology 12 (2005) 264-269.