Human ubiquitin-protein ligase NEDD4-like catalytic domain

Target ID:

NEDD4L

Aliases:

FLJ33870hNedd4-2KIAA0439RSP5

Deposition Date:

Wednesday 24th January 2007

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Related Diseases:

SignallingNeurobiology

Structure type:

apo

Download Coordinates:

2ONI

Authors:

J.R.WALKER,G.V.AVVAKUMOV,S.XUE,C.BUTLER-COLE,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

2ONI

tabs

Structure Details

The E3 ligase Nedd4l regulates levels of the kidney epithelial sodium channel by affecting the channelâ€™s rate of degradation. Ubiquitylation of the protein leads to endocytosis followed by degradation in lysosomes. The ligase interacts with the channel through a WW domain that recognizes a polyproline motif in the channel. This activity of Nedd4l is in turn regulated by the glucocorticoid-inducible kinase, which phosphorylates residues necessary for nedd4l-channel interaction. Notably, mutations in nedd4l correlate with susceptibility to hypertension[1]. Nedd4l is therefore an important component in the reabsorption of sodium in the kidneys[2].
Nedd4l is a member of hect-containing ubiquitin ligases that also contain WW and C2 domains. This group of ligases differ from others in that a ubiquityl-ligase intermediate is formed before transfer to substrate. Previously-determined structures show significant flexibility in the relative orientation between amino- and carboxy-terminal domains, which is assumed to be necessary for the transfer of the ubiquityl moiety from E2 to substrate. We have determined the high resolution structure of the hect domain of Nedd4l, revealing the canonical bilobal architecture. The short distance between the active site cysteine of the hect domain and the putative position of the E2 active site cysteine provides further insight into the mechanism of action.

Materials and Methods

NEDD4L

PDB:2ONI
Entry Clone Accession:gi:21619659
Entry Clone Source:nedd4l.BC032597.MGC.AT55-F3.pCMV-SPORT6
SGC Clone Accession:
Tag:mhhhhhhssgrenlyfqG
Host:BL21 (DE3)
Vector:p28a-LIC-TEV

Sequence:mhhhhhhssgrenlyfqGSREFKQKYDYFRKKLKKPADIPNRFEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKGLDYGGVAREWFFLLSKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAVFHGKLLDGFFIRPFYKMMLGKQITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQTYQVDLKPNGSEIMVTNENKREYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELELLMCGLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQLFTIEQWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVEN

Growth

Medium:Terrific Broth (TB)
Procedure: NEDD4L was 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. To produce SeMet derivative of NEDD4L, the same bacterial cells were grown using M9 SeMet High-Yield growth media kit package (Medicilon) according to manufacturer's instructions.

Purification

Procedure: 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. The protein was 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 10 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.

Structure Determination

Crystallization:Crystals were grown in hanging drops by mixing 2 microL protein solution with 2 microL well solution (1.7 M sodium/potassium phosphate, pH 6.0, 1 mM DTT) at 21 degC. For cryoprotection, the crystals were soaked in 2 M sodium-potassium phosphate, pH 7.0, 1 mM DTT, 25% ethylene glycol and 2 mg/ml Nedd4l.574.947.

References

Fouladkou,F. et al. A naturally occurring human Nedd4-2 variant displays impaired ENaC regulation in Xenopus laevis oocytes. Am. J. Physiol Renal Physiol 287, F550-F561 (2004).
Snyder,P.M. Minireview: regulation of epithelial Na+ channel trafficking. Endocrinology 146, 5079-5085 (2005)