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