Structural basis for DNA recognition and allosteric control of the retinoic acid receptors RAR–RXR

Osz J, McEwen A, Bourguet M, Przybilla F, Peluso-Iltis C, Poussin-Courmontagne P, Mély Y, Cianférani S, Jeffries C, Svergun D, Rochel N, Nucleic Acids Research (2020) DOI

SASDFT8 – The retinoic acid receptor (RAR-RXR heterodimer) bound to the DNA response element HoxB13 DR0

Retinoic acid receptor alpha, RAR
Retinoic acid receptor RXR-alpha
DNA response element HoxB13 DR0
MWexperimental 103 kDa
MWexpected 88 kDa
VPorod 132 nm3
log I(s) 8.09×101 8.09×100 8.09×10-1 8.09×10-2
Retinoic acid receptor alpha, RAR Retinoic acid receptor RXR-alpha DNA response element HoxB13 DR0 small angle scattering data  s, nm-1
ln I(s)
Retinoic acid receptor alpha, RAR Retinoic acid receptor RXR-alpha DNA response element HoxB13 DR0 Guinier plot ln 8.10×101 Rg: 3.8 nm 0 (3.8 nm)-2 s2
(sRg)2I(s)/I(0)
Retinoic acid receptor alpha, RAR Retinoic acid receptor RXR-alpha DNA response element HoxB13 DR0 Kratky plot 1.104 0 3 sRg
p(r)
Retinoic acid receptor alpha, RAR Retinoic acid receptor RXR-alpha DNA response element HoxB13 DR0 pair distance distribution function Rg: 4.0 nm 0 Dmax: 14.5 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Retinoic acid receptor alpha, RAR Retinoic acid receptor RXR-alpha DNA response element HoxB13 DR0 CORAL model

Synchrotron SAXS data from solutions of the retinoic acid receptor (RXR/RAR heterodimer) bound to the DNA response element HoxB13 DR0 in 20 mM Tris, pH 8, 150 mM NaCl, 5% v/v glycerol, 1 mM CHAPS, 4 mM MgSO4, 1 mM TCEP were collected on the EMBL P12 beam line at PETRA III (Hamburg, Germany) using a Pilatus 2M 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 100.00 μl sample at 5.5 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 10/300 column at 20°C. 45 successive 1 second frames were collected through the SEC-elution sample peak. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

SEC-SAXS analysis was performed in parallel with right-angle laser light scattering (RALLS) and refractive index measurements at room temperature. The Rg-correlation through the SEC-SAXS peak, as well as the results from coupled RALLS/RI, and alternative CORAL rigid-body refined models that fit the scattering data are included in the full entry zip archive. The quoted experimental molecular weight was determined from RALLS/RI measurements that were recorded from the same sample eluting from the column using a split-flow SEC-SAXS-light scattering configuration as described in Graewert et al., (2015) Sci. Reports. 5, 10734: doi: 10.1038/srep10734. The MW estimated directly from the SAXS data using Bayesian inference is ca. 86 kDa in the interval 81-93 kDa (Hajizadeh et al., (2018) Sci. Reports. 8(1):7204. doi: 10.1038/s41598-018-25355-2).

Retinoic acid receptor alpha, RAR (mRARa)
Mol. type   Protein
Organism   Mus musculus
Olig. state   Monomer
Mon. MW   40.8 kDa
 
UniProt   P11416 (84-421)
Sequence   FASTA
 
Retinoic acid receptor RXR-alpha (mRXRa)
Mol. type   Protein
Organism   Mus musculus
Olig. state   Monomer
Mon. MW   37.7 kDa
 
UniProt   P28700 (135-467)
Sequence   FASTA
 
DNA response element HoxB13 DR0 (HoxB13 DR0)
Mol. type   DNA
Olig. state   Monomer
Mon. MW   9.9 kDa
Sequence   FASTA