Molecular basis for dimer formation of TRβ variant D355R

Abstract

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 Å resolution with Rfree/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by a two-fold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the Cterminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 Å 2 characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.

Structure of the homodimeric androgen receptor ligand-binding domain

“…Finally, H5-centred symmetric dimers might be additionally relevant for other NRs. In this regard, previously reported structures of TR homodimers reveal that topologically equivalent elements from H5 and L5–6 contribute to the dimer interface 51 52 ( Supplementary Fig. 5c ).…”

Non-canonical dimerization of the androgen receptor and other nuclear receptors: implications for human disease

“…Whether are least some of these variants would impair dimer formation has not been experimentally confirmed. However, inspection of the 3D structure of the TRβ homodimer (Estébanez-Perpiñá et al 2007a, Jouravel et al 2009 immediately reveals that some of the reported missense mutations actually map to the homodimer interface and are therefore likely to disrupt receptor homodimerization (Fig. 5D).…”

The oligomeric state of thyroid receptor regulates hormone binding kinetics

“…Furthermore, HD exchange experiments (Figueira et al 2011) confirm that hormone binding results in remodeling of the dimer surface. We emphasize that enhanced dimer formation by the TRb D355R mutant involves novel surfaces in the H10-H11 loop and H8-H6 that lie close to the classic dimer surface at H10-H11 but are not identical to this interaction surface (Jouravel et al 2009). The fact that dimer formation through an unusual surface affects hormone binding kinetics in a similar manner to native dimer formation raises the possibility that effects of dimerization on ligand binding kinetics may be related partly to physical occlusion of ligand entry/exit routes as well as allosteric connections between the LBP and the classic dimer face.…”

“…TRbD355R exhibits enhanced dimer formation on DNA and in solution, as judged by non-denaturing gel electrophoresis and sizing columns (Togashi et al 2005b, Jouravel et al 2009 and not shown). Analysis of association rates revealed that TRbD355R bound T 3 with single-phase kinetics characterized by slow on rate (K on T 1/2 Z0 .…”

“…We have been unable to obtain purified wild-type TR dimers devoid of monomers, so we examined T 3 association with a TR mutant (TRbD355R) that exhibits enhanced dimer formation on DNA in the presence of T 3 (Togashi et al 2005b) and uses an unusual dimer interface that comprises the loop between H10 and H11, near the typical dimer interface at the H10-H11 junction, with auxiliary mutant-specific contacts between H8 and H6 (Jouravel et al 2009). …”