Transient interdomain interactions in free USP14 shape its conformational ensemble.

Salomonsson J, Wallner B, Sjöstrand L, D'Arcy P, Sunnerhagen M, Ahlner A, Protein Sci 33(5):e4975 (2024) Europe PMC

SASDQZ7 – Ubiquitin-specific protease 14 (USP14)

Ubiquitin carboxyl-terminal hydrolase 14
MWexperimental 56 kDa
MWexpected 56 kDa
VPorod 111 nm3
log I(s) 1.06×103 1.06×102 1.06×101 1.06×100
Ubiquitin carboxyl-terminal hydrolase 14 small angle scattering data  s, nm-1
ln I(s)
Ubiquitin carboxyl-terminal hydrolase 14 Guinier plot ln 1.06×103 Rg: 3.1 nm 0 (3.1 nm)-2 s2
(sRg)2I(s)/I(0)
Ubiquitin carboxyl-terminal hydrolase 14 Kratky plot 1.104 0 √3 sRg
p(r)
Ubiquitin carboxyl-terminal hydrolase 14 pair distance distribution function Rg: 3.3 nm 0 Dmax: 12.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Ubiquitin carboxyl-terminal hydrolase 14 OTHER [STATIC IMAGE] model
log I(s)
 s, nm-1
Ubiquitin carboxyl-terminal hydrolase 14 DAMMIN model
Synchrotron SAXS data from solutions of Ubiquitin-specific protease 14 (USP14) in 20 mM HEPES, 150 mM NaCl, 2 mM TCEP, 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 = 0.124 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 75.00 μl sample at 12 mg/ml was injected at a 0.60 ml/min flow rate onto a GE Superdex 75 Increase 10/300 column at 20°C. 2400 successive 1 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.

SEC-SAXS measurements on full-length USP14. The experimental molecular weight was determined from SEC-MALLS run in parallel to SEC-SAXS. Structural sampling of the two domains of UPS14, and the generation of a refined ensemble of structural states that fit the SAXS data (displayed, bottom) were calculated using the iterative Bayesian/Maximum Entropy approach (iBME) as described in Pesce F, Lindorff-Larsen K (2021) Refining conformational ensembles of flexible proteins against small-angle x-ray scattering data. Biophys J 120:5124–5135. The results from iBME are included in the full entry zip archive.

Ubiquitin carboxyl-terminal hydrolase 14 (USP14)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   56.2 kDa
 
UniProt   P54578 (1-494)
Sequence   FASTA