Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

Sacerdoti M, Gross L, Riley A, Zehnder K, Ghode A, Klinke S, Anand G, Paris K, Winkel A, Herbrand A, Godage H, Cozier G, Süß E, Schulze J, Pastor-Flores D, Bollini M, Cappellari M, Svergun D, Gräwert M, Aramendia P, Leroux A, Potter B, Camacho C, Biondi R, Science Signaling 16(789) (2023) DOI

SASDNK5 – Full length 3-phosphoinositide-dependent protein kinase (PDK1) in the presence of HYG8 (2-O-benzoyl-Ins(1,3,4,5,6)P5)

3-phosphoinositide-dependent protein kinase 1
2-O-benzoyl-Ins(1,3,4,5,6)P5
MWexperimental 54 kDa
MWexpected 66 kDa
VPorod 98 nm3
log I(s) 2.28×103 2.28×102 2.28×101 2.28×100
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 small angle scattering data  s, nm-1
ln I(s)
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 Guinier plot ln 2.29×103 Rg: 3.5 nm 0 (3.5 nm)-2 s2
(sRg)2I(s)/I(0)
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 Kratky plot 1.104 0 √3 sRg
p(r)
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 pair distance distribution function Rg: 3.5 nm 0 Dmax: 11.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 DAMMIF model
log I(s)
 s, nm-1
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 EOM/RANCH model
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 EOM/RANCH model
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 EOM/RANCH model
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 EOM/RANCH model
3-phosphoinositide-dependent protein kinase 1 2-O-benzoyl-Ins(1,3,4,5,6)P5 EOM/RANCH model
Synchrotron SAXS data from solutions of full length PDK1 in the presence of HYG8 in 20 mM Tris-HCl pH 7.4, 250 mM NaCl, 1 mM DTT, 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 10.00 μl sample at 6 mg/ml was injected at a 0.30 ml/min flow rate onto a GE Superdex 200 Increase 5/150 column at 20°C. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC-SAXS run from a sample of full length PDK1 at 6 mg/ml (100 μM) pre-incubated with Hyg8 at 500 μM. 1 μM Hyg8 was also added to the SEC running buffer.

3-phosphoinositide-dependent protein kinase 1 (PDK1 FL)
Mol. type   Protein
Organism   Homo sapiens
Olig. state   Monomer
Mon. MW   64.9 kDa
 
UniProt   O15530 (2-556)
Sequence   FASTA
 
2-O-benzoyl-Ins(1,3,4,5,6)P5 (HYG8)
Mol. type   Other
Organism   synthetic construct
Olig. state   Monomer
Mon. MW   0.6 kDa