PDBsum entry 1xap

Transcription

PDB id: 1xap

Contents

Protein chain

232 a.a. *

Ligands

TTB

Waters ×115

* Residue conservation analysis

PDB id:

1xap

Name:

Transcription

Title:

Structure of the ligand binding domain of the retinoic acid receptor beta

Structure:

Retinoic acid receptor beta. Chain: a. Fragment: ligand binding domain. Synonym: rar-beta, rar-epsilon, hbv-activated protein. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.10Å R-factor: 0.213 R-free: 0.253

Authors:

P.Germain,S.Kammerer,C.Peluso-Iltis,D.Tortolani,F.C.Zusi,J.Starrett, P.Lapointe,J.P.Daris,A.Marinier,A.R.De Lera,N.Rochel,H.Gronemeyer

Key ref:

P.Germain et al. (2004). Rational design of RAR-selective ligands revealed by RARbeta crystal stucture. EMBO Rep, 5, 877-882. PubMed id: 15319780 DOI: 10.1038/sj.embor.7400235

Date:

26-Aug-04 Release date: 16-Nov-04

Protein chain

P10826  (RARB_HUMAN) -  Retinoic acid receptor beta from Homo sapiens

Seq: 455 a.a.

Struc: 232 a.a.

DOI no: 10.1038/sj.embor.7400235

EMBO Rep 5:877-882 (2004)

PubMed id: 15319780

Rational design of RAR-selective ligands revealed by RARbeta crystal stucture.

P.Germain, S.Kammerer, E.Pérez, C.Peluso-Iltis, D.Tortolani, F.C.Zusi, J.Starrett, P.Lapointe, J.P.Daris, A.Marinier, A.R.de Lera, N.Rochel, H.Gronemeyer.

ABSTRACT

The crystal structure of the ligand-binding domain of RARbeta, a suspect tumour suppressor, reveals important features that distinguish it from the two other RAR isotypes. The most striking difference is an extra cavity allowing RARbeta to bind more bulky agonists. Accordingly, we identified a ligand that shows RARbeta selectivity with a 100-fold higher affinity to RARbeta than to alpha or gamma isotypes. The structural differences between the three RAR ligand-binding pockets revealed a rationale explaining how a single retinoid can be at the same time an RARalpha, gamma antagonist and an RARbeta agonist. In addition, we demonstrate how to generate an RARbeta antagonist by gradually modifying the bulkiness of a single substitution. Together, our results provide structural guidelines for the synthesis of RARbeta-selective agonists and antagonists, allowing for the first time to address pharmacologically the tumour suppressor role of RARbeta in vitro and in animal models.

Selected figure(s)

Figure 1.
Figure 1 Crystal structure of the RAR LBD -TTNPB complex reveals an additional cavity in the RAR LBP. (A) Electron density map of TTNPB in its pocket. 2F[o] -F[c] map at 2.1 � resolution contoured at 1 . The three isotype-specific residues (A[225], I[263] and V[388]) and residues anchoring the carboxylate (R[269] and S[280]) are indicated. Illustration by PYMOL. (B) Superposition of the holo-RAR -TTNPB (yellow) and RAR -9-cis RA (blue) LBDs. Illustration by SETOR. (C) Superposition of TTNPB -RAR (blue) and 9-cis RA -RAR (grey) LBDs. The isotype-specific residues are shown in cyan (RAR ) and orange (RAR ) and TTNPB in yellow. The carboxylate anchoring residues are illustrated as ball-and-sticks. H bonds are represented as dashed lines. The figure was prepared by MOLSCRIPT and RASTER3D. (D) Superimposition of RAR (blue) and RAR (grey) LBPs. The arrow points to the additional cavity in RAR .

Figure 5.
Figure 5 The three divergent LBP residues determine the RAR selectivity of BMS641 and the isotype-dependent potential of BMS453. (A) Partial proteolysis maps of in vitro-translated RARs in the presence or absence of increasing concentrations of either TTNPB, BMS453 or BMS641, as indicated. (B) Dose -response curves to assess the binding affinity of BMS641 relative to TTNPB in RAR pocket mutants. HeLa cells were co-transfected with (RARE)3x-tk-luc and either RAR (closed triangles), RAR (open circles), RAR arrow (RAR (M[272]I, A[397]V); open squares) or RAR arrow (RAR (I[263]M, V[388]A); closed diamonds) and reporter gene transcription was induced with 3 nM TTNPB (100%). (C) BMS453-induced luciferase activity in HeLa cells co-transfected with (RARE)3x-tk-luc and the indicated receptors.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: EMBO Rep (2004, 5, 877-882) copyright 2004.

Figures were selected by the author.

Author's comment

Together with our previous studies structural features can be deduced for the synthesis of RAR isotype-selective retinoids, (i) the three divergent residues and the different shape of the RARβ LBP reported here are the most important discriminatory elements; (ii) existing isotype-selective ligands dissociate mostly RARα from RARβ/γ by exploiting the presence of RARαS232, which can establish hydrogen bonds with suitable ligands; we predicted this earlier for ligands harboring an amino group, such as Am580 or BMS753; (iii) to separate RARβ from RARα binding a 3-chloro substitution that appears to create steric hindrance within the RARα pocket can be introduced in TTNPB-like retinoids; (iv) to separate RARβ from RARγ binding two structural aspects can be exploited, both of which are a consequence of the replacement of RARβI263 by RARγM272. First, the side chain orientation of I263 opens a cavity that is closed in RARγ; ligands that require such a cavity to accommodate a bulky substituent acquire β-selectivity. Our functional analyses have shown that a 3-chloro (in UVI2007) or a 8”-phenyl (in BMS453) on their own can not discriminate between RARβ and RARγ; however, the combination of both substitutions (in BMS641) results despite an overall lower affinity in acquisition of β-selective binding that originates from a dramatic loss of RARγ affinity. This is highly suggestive of a structural model in which the 3-substitution positions the ligand in the RARγ pocket such that the 8’’-phenyl clashes with M272 (but not the corresponding I263 of RARβ). Second, the presence of M272 allows establishing hydrogen bonds to a suitable ligand, e.g., BMS270394, to augment γ affinity and selectivity; (v) in all cases antagonists are characterized by the presence of substitutions that interfere with the holo conformation of H12. Hinrich Gronemeyer