Structural basis of ubiquitin ligase Nedd4-2 autoinhibition and regulation by calcium and 14-3-3 proteins

Janosev M, Kosek D, Tekel A, Joshi R, Honzejkova K, Pohl P, Obsil T, Obsilova V, Nature Communications 16(1) (2025) DOI

SASDUC3 – Phosphorylated E3 ubiquitin-protein ligase Nedd4-2 in the absence of calcium

Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like
MWexperimental 119 kDa
MWexpected 110 kDa
VPorod 206 nm3
log I(s) 9.82×10-2 9.82×10-3 9.82×10-4 9.82×10-5
Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like small angle scattering data  s, nm-1
ln I(s)
Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like Guinier plot ln 9.83×10-2 Rg: 4.9 nm 0 (4.9 nm)-2 s2
(sRg)2I(s)/I(0)
Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like Kratky plot 1.104 0 √3 sRg
p(r)
Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like pair distance distribution function Rg: 5.2 nm 0 Dmax: 22 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like CORAL model
Synchrotron SAXS data from solutions of phosphorylated E3 ubiquitin-protein ligase Nedd4-2 in 20 mM HEPES, 150 mM NaCl, 1 mM TCEP, 1 mM EDTA, 3% glycerol, pH 7.5 were collected on the EMBL P12 beam line at PETRA III (DESY; Hamburg, Germany) using a Pilatus 6M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.123982 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 8.3 mg/ml was injected at a 0.30 ml/min flow rate onto a GE Superdex 200 Increase 5/150 column at 20°C. 1800 successive 0.495 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Isoform 5 of E3 ubiquitin-protein ligase NEDD4-like (Nedd4-2 iso5 human)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   110.3 kDa
 
UniProt   Q96PU5-5 (1-955)
Sequence   FASTA