Human ubiquitin protein ligase NEDD4-like

Target ID:

NEDD4L

Aliases:

FLJ33870hNedd4-2KIAA0439RSP5

Deposition Date:

Monday 6th November 2006

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2NSQ

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,C.BUTLER-COLE,P.J.FINERTYJR.,J.WEIGELT,M.SUNDSTROM,C.H.ARROWSMITH,A.M.EDWARDS,A.BOCHKAREV,S.DHE-PAGANON,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Toronto

2NSQ

tabs

Structure Details

Liddle syndrome, cystic fibrosis, primary erythermalgia, and hypokalaemic periodic paralysis are pathologies that result from disregulation of their associated ion channels: Scnn1b, Cftr, Scn9a, And Cacnl1a3, respectively. Nedd4l is a Hect-E3 ligase that has been shown to regulate the degradation of the epithelial sodium channel (EnaC/Scnn1), and is thought to regulate the surface expression of the voltage-gated sodium channel Scn9a, which is involved in pain reception. By targeting the epithelial sodium channel for degradation, Nedd4l is a significant determinant of sodium reabsorption in the distal nephron. Genetic linkage to hypertension has been reported to a region of chromosome 18q harboring the gene, with phenotypes that include a rare orthostatic hypotension disorder, essential hypertension, and postural change in systolic blood pressure. The ubiquitin ligase NEDD4L is therefore a candidate gene for essential hypertension on both functional and genetic grounds.

The Nedd4 family of E3 ubiquitin ligases share a common domain architecture including a C-terminal catalytic Hect domain, WW domains, and an N-terminal C2 domain. The Hect domain is responsible for transferring ubiquitin from an E2 to substrate, while the WW domains are responsible for substrate recognition. In the case of Liddleâ€™s syndrome, mutations in the epithelial sodium channel prevent it from interacting with the WW domain of Nedd4l. The C2 domain, a phospholipid binding domain, aids in target recognition by directing the ligase to channel-containing membranes. In order to further elucidate the function of the Nedd4l E3 ligase, we have determined the high-resolution crystal structure of its C2 domain.

Materials and Methods

NEDD4L C2 domain

PDB:2NSQ
SGC Clone Accession:nedd4l.0001.0154; plate SDC087, well D8

Sequence:gMATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLYVADENRELALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLTRDDFLGQVDVPLSHLPTEDPTMERPYTFKDFLLRPRSHKSRVKGFLRLKMAYMP

Growth

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

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 column from BD Biosciences at 4 degC. 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, 2mM dithiothreitol and concentrated by ultrafiltration.

Extraction

Procedure: The cell pellet was resuspended in lysis buffer inhibitor (0.1mM phenylmethyl sulfonyl fluoride, PMSF) and lysed using Microfluidizer. The lysate was cleared by centrifugation.

Structure Determination

Crystallization:Crystals of NEDD4L-C2 were obtained by means of hanging drop vapor diffusion in 14% PEG 4000, 0.2 M NaOAc, pH 6.5 in room temperature (298K),with 1 mM DTT added as reducing agent and 20% ethylene glycol added as cryo protector.