Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2.

Abdul Rehman SA, Armstrong LA, Lange SM, Kristariyanto YA, Gräwert TW, Knebel A, Svergun DI, Kulathu Y, Mol Cell (2021) Europe PMC

SASDJB3 – Ubiquitin carboxyl-terminal hydrolase, MINDY2 (C266A mutant), bound to di-ubiquitin

Ubiquitin carboxyl-terminal hydrolase C266A mutant
Polyubiquitin-C
MWexperimental 43 kDa
MWexpected 48 kDa
VPorod 68 nm3
log I(s) 1.42×103 1.42×102 1.42×101 1.42×100
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C small angle scattering data  s, nm-1
ln I(s)
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C Guinier plot ln 1.42×103 Rg: 2.5 nm 0 (2.5 nm)-2 s2
(sRg)2I(s)/I(0)
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C Kratky plot 1.104 0 √3 sRg
p(r)
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C pair distance distribution function Rg: 2.5 nm 0 Dmax: 8 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C CUSTOM IN-HOUSE model
log I(s)
 s, nm-1
Ubiquitin carboxyl-terminal hydrolase C266A mutant Polyubiquitin-C CUSTOM IN-HOUSE model
Synchrotron SAXS data from solutions of MINDY2 (C266A mutant) bound to di-ubiquitin in 20 mM HEPES, 100 mM NaCl, 5 mM DTT, 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 = 0.12 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 9.9 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 3000 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.

The experimental MW was determined from Porod volume.

Ubiquitin carboxyl-terminal hydrolase C266A mutant (MINDY-2 C266A)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   31.0 kDa
 
UniProt   Q8NBR6 (241-504)
Sequence   FASTA
 
Polyubiquitin-C (UBC - dimer)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Dimer
Mon. MW   8.6 kDa
 
UniProt   P0CG48 (1-76)
Sequence   FASTA