Human hect domain and RLD 2, third RLD domain

Target ID:

HERC2

Deposition Date:

Wednesday 21st October 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

apo

Download Coordinates:

3KCI

Authors:

Walker JR, Qiu L, Vesterberg A, Weigelt J, Bountra C, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

3KCI

tabs

Structure Details

The molecular basis of human pigmentation is a fascinating topic but not well understood. Recent studies show that it is a polygenic trait involving the ubiquitylation system. The interaction between HERC2 and OCA2, for example, may be responsible for the blue eye color in humans, and association between HERC2 and MC1R may play a role in determining skin and hair color. HERC2 is an E3 ligase with a canonical HECT domain at its carboxy terminus, three WD40-like β-propellers (here referred as RLD1, RLD2, and RLD3), and various other smaller domains including a cytochrome B5-like domain. To begin to understand how HERC2 contributes to human pigmentation, we are studying its biochemical interactions. We solved the structure of the third RLD domain of HERC2 and show that it adopts a seven-bladed β-propeller fold. The inner strand of each blade is aligned with the central water-filled cavity of the propeller. The amino- and carboxy-terminal tails lie next to each on the same side of the propeller. The diameter of its central cavity is significantly larger than other WD40-like proteins and may provide clues to the nature of potential ligands.

Materials and Methods

HERC2

PDB:3KCI
Entry Clone Accession:Herc2.LIFESEQ3382404.OBS.IHS1382-8549203.pINCY
Entry Clone Source:OpenBiosystems
SGC Clone Accession:herc2.3951.4321 (SDC174D02)
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Vector:pET28a-LIC

Sequence: mhhhhhhssgrenlyfqgSGTIYGWGHNHRGQLGGIEGAKVKVPTPCEALATLRPVQLIGGEQTLFAVTADGKLYATGYGAGGRLGIGGTESVSTPTLLESIQHVFIKKVAVNSGGKHCLALSSEGEVYSWGEAEDGKLGHGNRSPCDRPRVIESLRGIEVVDVAAGGAHSACVTAAGDLYTWGKGRYGRLGHSDSEDQLKPKLVEALQGHRVVDIACGSGDAQTLCLTDDDTVWSWGDGDYGKLGRGGSDGCKVPMKIDSLTGLGVVKVECGSQFSVALTKSGAVYTWGKGDYHRLGHGSDDHVRRPRQVQGLQGKKVIAIATGSLHCVCCTEDGEVYTWGDNDEGQLGDGTTNAIQRPRLVAALQGKKVNRVACGSAHTLAWSTSKP

Growth

Procedure: Competent BL-21(DE3) cells (Invitrogen, C6000-03) were transformed and grown using the LEX system (Harbinger BEC) at 37 degC in 2L bottles (VWR, 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 150 mM glycerol, 100 µM Kanamycin and 600 µl antifoam 204 (Sigma A-8311). At OD600 = 6, the temperature was reduced to 15 degC, and one hour later the culture was induced with 100 µM IPTG (BioShop IPT001) and incubated overnight (16 hours) at 15 degC. Cell pellets were collected by centrifugation (12,227 xg, 20 mins) and frozen in liquid nitrogen.

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. Resin was transferred to a column and washed with 5 column volumes (cv) of Wash buffer A, 5 cv of Wash buffer B, and 5 cv of Wash buffer A. The protein was eluted with 2 cv of Elution buffer. The protein was further purified by gel filtration through a HighLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by ABS280 nm) were pooled and concentrated to 0.5-1.0 mM using concentrators (Amicon) with 5 kDa cutoff. The yield of the protein was approximately 10 mg per L of bacterial culture. Coomassie-stained, SDS-PAGE showed that the product was pure and Mass-spectroscopy by LCMS (Agilent 1100 Series) showed that the protein has 107 Da more than the calculated molecular weight.

Extraction

Procedure:
Cell pellets were resuspended in Lysis buffer(30 mL per L culture), lysed using a Microfluidizer (Microfluidics, M110-EH) at 18,000 psi, and cleared by centrifugation (40,000 xg for 30 minutes).

Structure Determination

Crystallization:Crystals were grown at 18 oC using the sitting drop method in 96well plates (Art Robbins, 102-0004-00) by mixing equal volumes of protein (8.0 mg/ml) and Crystallization Buffer (20% PEG3350, 0.2mM MgCl2). Suitable crystals were cryoprotected by immersion in well solution supplemented with 25% (v/v) Glycerol prior to dunking and storage in liquid nitrogen.
Data Collection:Diffraction data from a crystal of the third RCC1-like domain of HERC2 was collected on a home source Rigaku FR-E, and integrated and scaled using the HKL2000 program suite.
Data Processing:The structure was solved by molecular replacement techniques using the program PHASER and search model PDB entry 1A12. Automated model building using ARP/wARP, combined with iterative model building using the graphics program Coot and maximum-likelihood and TLS refinement with the program REFMAC5 led to a model with an R factor of 15.2% (Rfree 19.5%) for data between 20.0-1.8 Å. Parameters for Translation/liberation/screw (TLS) refinement were generated using the TLSMD web server.