Human HECT, UBA and WWE domain containing 1

Target ID:

HUWE1

Deposition Date:

Friday 30th January 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

apo

Download Coordinates:

3G1N

Authors:

Walker JR, Qiu L, Li Y, Tempel W, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Botchkarev A, Dhe-Paganon S

Site:

Toronto

3G1N

tabs

Structure Details

Human Huwe1 (also called Mule or ARF-BP1) is a UBA- and WWE-containing HECT E3 ubiquitin ligase that is expressed in many tissue types[1] and is found predominantly in the cytoplasm[2]. It is implicated in the ubiquitylation and subsequent proteasomal degradation of a number of targets, including MYCN, MCL1, p53, CDC6, and Histones. By degrading these targets, Huwe1 promotes proliferation and inhibit apoptosis.

Huwe1 is composed of the canonical HECT fold, consisting of 2 major lobes, the N-terminal fold, which recruits the loaded E2 cofactor, and the C-terminal lobe, which contains the catalytic cysteine (which forms the thiol-ester intermediate). There are at least two important transition states in reactions catalyzed by HECT enzymes, the first characterized by the transfer of ubiquitin from E2 to the catalytic HECT cysteine, and the second characterized by the transfer of ubiquitin from enzyme to substrate. Both transitions are poorly characterized. Crystal structures to date have shown significant flexibility between the two major lobes, presumably important because E2 and substrate binding sites are likely non-overlapping; the position of the E2-binding pocket is known, but that of the substrate is not.

The Huwe1 structure shows that the orientation between major lobes is similar to that of WWP1 (PDB 1ND7), where the catalytic cysteine is about 20 Angstroms from the putative catalytic E2 cysteine. That this orientation is conserved suggests functional relevance. The Huwe1 structure also reveals a unique conformation of the β5-β6 loop (4083-89) in the major, N-terminal lobe that appears to determine the position of the major, C-terminal lobe relative to the putative E2 binding pocket. Notably, the sidechain carboxyl of Asn4087 in this loop forms a hydrogen bond with a conserved sodium atom situated behind the catalytic cysteine.

Materials and Methods

HUWE1

PDB:3G1N
Entry Clone Accession:NP_113584
Entry Clone Source:huwe1.NM_031407.LAB.Martin_Eilers_dnd.pCMVHA
SGC Clone Accession:huwe1.4006.4374.158A03 (SDC158A03)
Tag:N-terminal: MHHHHHHSSGRENLYFQG
Host:BL-21(DE3)V2R
Vector:pET28-MHL

Sequence: mhhhhhhssgrenlyfqgLERLDEGLRKEDMAVHVRRDHVFEDSYRELHRKSPEEMKNRLYIVFEGEEGQDAGGLLREWYMIISREMFNPMYALFRTSPGDRVTYTINPSSHCNPNHLSYFKFVGRIVAKAVYDNRLLECYFTRSFYKHILGKSVRYTDMESEDYHFYQGLVYLLENDVSTLGYDLTFSTEVQEFGVCEVRDLKPNGANILVTEENKKEYVHLVCQMRMTGAIRKQLAAFLEGFYEIIPKRLISIFTEQELELLISGLPTIDIDDLKSNTEYHKYQSNSIQIQWFWRALRSFDQADRAKFLQFVTGTSKVPLQGFAALEGMNGIQKFQIHRDDRSTDRLPSAHTCFNQLDLPAYESFEKLRHMLLLAIQECSEGFGLA

Growth

Medium:TB
Procedure: A volume of 1.8 L of TB (Sigma T0918) supplemented with glycerol (final conc 100 µM), Kanamycin (final conc 85 µM), and antifoam 204 (Sigma A-8311; about 600 µL) was inoculated with 50 mL of overnight LB pre-culture and aerated with the LEX system at 37 °C. When the OD600 6, the temperature of the media was reduced to 15 °C (which required about 1 hour) and the culture was induced with 100 µM IPGT (BioShopCanada IPT001). Cultures were aerated overnight (16 hours) at 15 °C, and cell pellets were collected by centrifugation and frozen in liquid nitrogen before storage.

Purification

Procedure: Cleared lysate was rocked with TALON metal-affinity resin (BD Biosciences; 1.5 mL settled beads per L cell culture) at 4 °C. The column was washed with 5 column volumes of Wash buffer A, 5 column volumes of Wash buffer B, and 5 column volumes of Wash buffer A. The protein was eluted with 2 column volumes of Elution buffer.

The protein was further purified by gel filtration through a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by ABS280 nm) were pooled and concentrated by ultra filtration using an Amicon Ultra centrifugal filter with 5 kDa cutoff.

The yield of the protein was approximately 2 mg per L of bacterial culture.

Extraction

Procedure: Cell pellets were resuspended in Lysis buffer (30 mL per L culture), lysed using a Microfluidizer (18,000 psi), and cleared by centrifugation (40,000 xg for 30 minutes).

Structure Determination

MassSpec:Mass-spectroscopy by LCMS showed that the product was pure and had the correct molecular weight .
Crystallization:Crystals were grown at 20 °C using the hanging drop method by mixing 2 volumes of the protein (20 mg/mL in 20 mM TrispH8.0, 300 mM NaCl, 0.5 mM TCEP) with 1 volume of well solution consisting of 15% PEG-4000, 10% iso-propanol, 0.1 M Tris-pH7.5. Crystals were cryoprotected by emersion in well solution supplemented with 50% (volume/volume) paratone before frozen in liquid nitrogen.

References

Chen, D. et al. ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor. Cell 121, 1071-83 (2005).
Liu, Z., Miao, D., Xia, Q., Hermo, L. & Wing, S.S. Regulated expression of the ubiquitin protein ligase, E3(Histone)/LASU1/Mule/ARF-BP1/HUWE1, during spermatogenesis. Dev Dyn 236, 2889-98 (2007).