Biology Reference
In-Depth Information
to their DNA response elements fully validated the studies on the isolated LBDs
(
Chandra et al., 2008, 2013
). Indeed, both the general dimeric organization and the
details of the inter-LBD interactions observed in the LBD dimers are strictly conserved
in the context of the full-length receptors. A notable exception to this conserved di-
meric arrangement is the case of 3-keto-steroid receptors (namely, the androgene
AR, progesterone (PR (NR3C3)), glucocorticoid (GR (NR3C1)), and mineralocorti-
coid (MR (NR3C2)) receptors) whose dimerization surface is partly masked by a
C-terminal extension folding as a
b
-strand and forming a
b
-sheet interaction with res-
idues located between helices H8 and H9 (
Fig. 2.2
C). The 3D structure of the GR LBD
homodimer (
Bledsoe et al., 2002
) suggested an alternative mode of dimerization in-
volving residues from a
b
-turn located between H5 and H6 and the extended loop be-
tween H1 and H3, as well as the last residue of H5 (
Fig. 2.2
C). However, formation of
the GR homodimer buries only 600
˚
2
of solvent-accessible surface as compared with
the 1000-1700
˚
2
of buried surfaces observed in the other NRs so that the biological
relevance of this mode of dimerization remains to be confirmed.
Considering that the methods used for the crystallization of protein complexes
are diverse and that crystallization conditions are unique to each protein (or protein
complex), we provide here a very general procedure that may apply to all kinds
of NR LBDs in complex or in isolation. The following protocol is an example
given for the crystallization of the RAR
a
-RXR
a
LBD heterodimers bound to
BMS614, a RAR
a
-selective antagonist.
2.1.1.1
Required materials
- Crystallization robot (X8 nanosystem, Cartesian; Freedom EVO, Tecan)
- Crystallization screens (Molecular Dimension, Hampton Research, etc.)
- 96-Well crystallization plates (Greiner Bio-One)
- 24-Well crystallization plates (Greiner Bio-One, Molecular Dimension)
- Siliconized glass coverslips (Greiner Bio-One)
- Stereo microscope (Leica)
- X-ray generator (Rigaku HF 007) with a MAResearch image plate
2.1.1.2
Protocol
Prior to crystallization trials, the purified heterodimer (see the succeeding text for
the description of a general purification method of NR heterodimers) is mixed with
threefold molar excess of the ligand, concentrated to 5-10 mg/mL, and centrifuged
for 30 min at 13,000 rpm to get rid of any aggregated material. An initial search for
crystallization conditions is undertaken by mixing the purified protein with sparse
matrix screening conditions using crystallization robots and dedicated 96-well
crystallization plates. This setup allows for the rapid screening of hundreds of crys-
tallization conditions commercially available in a 96-well format with a reduced
amount of protein. The robot automatically performs each crystallization trial
by mixing 0.1
m
L of protein with 0.1
m
L of precipitant condition in a well sus-
pended over a reservoir containing 100
m
L of the corresponding condition. Once
the 96 conditions have been dispensed, the plates are sealed with a transparent
