Ligand/cofactor complexes of nuclear receptor ligand-binding domains

Ligand/cofactor complexes of nuclear receptor ligand-binding domains H. Greschik, R. Flaig, and D. Moras Département de Biologie et Génomique Structurales, IGBMC, CNRS / INSERM / ULP, 1 rue Laurent Fries, B.P. 10142, 67404 Illkirch, France

Nuclear receptors (NRs) form a superfamily of ligand-regulated transcription factors that control diverse cellular events such as proliferation, differentiation, and homeostasis. NRs are multidomain proteins generally containing a N-terminal domain, a DNA-binding domain, and a Cterminal ligand-binding domain (LBD). The LBD adopts a so-called ‘α-helical sandwich fold’ consisting in general of 12 α-helices (H1-H12) and a short β-sheet, which are packed against one another in three layers [see Figure]. Ligand binding triggers significant conformational changes of the LBD. Most prominently, upon the binding of an agonist ligand, H12 folds back against the LBD body, seals the ligand-binding pocket, and - together with H3 and H4 - generates a novel, hydrophobic surface for the interaction with coactivators. Coactivators that specifically interact with agonist-bound LBDs include members of the p160-family (SRC1, TIF2, ACTR), CBP/p300, or the DRIP205 subunit of the Mediator complex. These coactivators contain LXXLL sequence motifs (L denotes leucine, X any amino acid) that mediate NR binding. The LXXLL-containing region forms an amphipathic α-helix [red ribbon in figure] that interacts with the hydrophobic cleft generated by H3, H4, and H12. Coactivator binding is further stabilized by a 'charge clamp' interaction with a conserved glutamic acid in H12 and a conserved lysin in H3 [reviewed in 1].

The described coactivator binding mode is conserved for most members of the NR superfamily, and it is currently unclear how specificity in NR LBD/cofactor interactions is achieved. Amino acids 5' or 3' of the LXXLL motif may contribute to specificity, however, crystal structures of a NR LBD complexed with a large coactivator domain have not yet been solved. Furthermore, distinct cofactors have been identified that appear to interact with NRs either via 'inverse' LXXLL motifs (LLXXL) or via unrelated sequences. For these cofactors the interaction mode in atomic detail is not known. Our project aims at determining crystal structures of NR LBDs with either unusual cofactor peptides or larger cofactor fragments (domains). We recently collected data sets for (i) a orphan NR LBD complexed with an ('inverse') LLXXL-containing peptide, and (ii) an orphan NR LBD in complex with a LXXLL-containing peptide, whose binding does not depend on the presence of the 'charge clamp' residues in H3 and H12. Both structures have been solved and refinement is in progress. Results are expected to contribute to the understanding of the determinants of NR LBD/cofactor binding specificity.

References [1] Greschik H. & Moras D. (2003). Structure-activity relationship of nuclear receptor-ligand interactions. Curr. Topics Med. Chem. 3: 1573-1599 (Review).