Title: Reevaluation of the PPAR-β/δ Ligand Binding Domain Model Reveals Why It Exhibits the Activated Form
Abstract: Peroxisome proliferator-activated receptors (PPARs), a subgroup of the nuclear hormone receptor family, adopt an activated configuration in the presence of an agonist (Nolte et al., 1998Nolte R.T. Wisely G.B. Westin S. Cobb J.E. Lambert M.H. Kurokawa R. Rosenfeld M.G. Willson T.M. Glass C.K. Milburn M.V. Nature. 1998; 395: 137-143Crossref PubMed Scopus (1607) Google Scholar) and bind the retinoid X receptor (RXR) and additional coactivator proteins to create a complex capable of interacting with specific DNA response elements. The target genes regulated by PPARs modulate lipid metabolism and/or glucose homeostasis together with energy expenditure (Willson et al., 2000Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1643) Google Scholar). PPAR-β/δ is the least understood of the three human PPAR isotypes, although it is implicated in lipid metabolism, cell survival, wound healing, embryonic implantation, and development of the central nervous system (Berger et al., 2005Berger J.P. Akiyama T.E. Meinke P.T. Trends Pharmacol. Sci. 2005; 26: 244-251Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, Willson et al., 2000Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1643) Google Scholar). Structural studies on the C-terminal ligand binding domain (LBD) of PPARs have been carried out (Li et al., 2003Li Y. Lambert M.H. Xu H.E. Structure. 2003; 11: 741-746Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Willson et al., 2000Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1643) Google Scholar). The PPAR-β/δ LBD is a single globular domain consisting of 12 α helices and a three-stranded β sheet (Xu et al., 1999Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Steinbach D.D. Lehman J.M. Wisely G.B. Willson T.M. et al.Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar) and closely resembles other nuclear receptors (Moras and Gronemeyer, 1998Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (677) Google Scholar). The elements of secondary structure create a large ligand binding cavity of approximate volume 1300 Å3. The LBD carries a C-terminal helix, termed the activation function helix-2 (AF-2), involved in coactivator binding. The position of AF-2 is determined by the presence, or absence, of an agonist in the LBD binding site (Molnár et al., 2005Molnár F. Matilainen M. Carlberg C. J. Biol. Chem. 2005; 280: 26543-26556Crossref PubMed Scopus (57) Google Scholar, Moras and Gronemeyer, 1998Moras D. Gronemeyer H. Curr. Opin. Cell Biol. 1998; 10: 384-391Crossref PubMed Scopus (677) Google Scholar). In PPAR-β/δ, the hydrogen bonding interactions, likely enhanced by the mainly hydrophobic environment of the binding cavity, formed by a buried fatty acid (FA) carboxylate and the AF-2 helix make important contributions to establishing a stable structure (Xu et al., 1999Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Steinbach D.D. Lehman J.M. Wisely G.B. Willson T.M. et al.Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar, Fyffe et al., 2006Fyffe S.A. Alphey M.S. Buetow B. Smith T.K. Ferguson M.A.J. Sørensen M.D. Björkling F. Hunter W.N. J. Mol. Biol. 2006; (in press)PubMed Google Scholar). A conundrum arising from previous work was the conclusion that the apo-PPAR-β/δ LBD displayed the same activated conformation as structures to which an FA or synthetic ligands had been complexed (Xu et al., 1999Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Steinbach D.D. Lehman J.M. Wisely G.B. Willson T.M. et al.Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). Our recent high-resolution crystallographic structures of recombinant human PPAR-LBD β/δ revealed a FA in the binding site, and mass spectrometry confirmed the presence of C16:0, C16:1, C18:0, and C18:1 in a ratio of approximately 3:2:1:4. Comparisons of the mean retention time differences between internal (C17:0) and various C18:1 standards led to the assignment of 11, Z octadecenoic acid (cis-vaccenic acid) as the most abundant C18:1 FA in the extract (Fyffe et al., 2006Fyffe S.A. Alphey M.S. Buetow B. Smith T.K. Ferguson M.A.J. Sørensen M.D. Björkling F. Hunter W.N. J. Mol. Biol. 2006; (in press)PubMed Google Scholar). These are endogenous FAs acquired from the bacterial expression system and serve to lock the LBD into the activated conformation. Because we used a similar expression vector/system to that reported previously, we decided to reassess the apo form of PPAR-β/δ LBD published in Molecular Cell (Protein Databank Bank accession code 2GWX, Xu et al., 1999Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Steinbach D.D. Lehman J.M. Wisely G.B. Willson T.M. et al.Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar). Diffraction data were retrieved from the PDB and molecular replacement methods (Vagin and Teplyakov, 2000Vagin A. Teplyakov A. Acta Crystallogr. 2000; D56: 1622-1624Google Scholar) were applied by using our improved high-resolution model, in which all water molecules and ligands had been removed, to position two molecules of the LBD (Fyffe et al., 2006Fyffe S.A. Alphey M.S. Buetow B. Smith T.K. Ferguson M.A.J. Sørensen M.D. Björkling F. Hunter W.N. J. Mol. Biol. 2006; (in press)PubMed Google Scholar). A different asymmetric unit was assigned, which resulted in the formation of more intimate interactions between the two molecules. One molecule retains position, and the other is related to that previously assigned by the symmetry operation 1 − x, y − 1/2, 1 − z. Refinement consisted of inspection of electron and difference density maps, and manipulation of models (Jones et al., 1991Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. 1991; A47: 110-119Crossref Scopus (12954) Google Scholar) interspersed with least squares optimization (Murshudov et al., 1999Murshudov G.N. Vagin A.A. Lebedev A. Wilson K.S. Dodson E.J. Acta Crystallogr. 1999; D55: 247-255Crossref Scopus (999) Google Scholar). Careful placement of water molecules and the identification of ligands completed the analyses. The initial electron density and difference density maps clearly indicated the presence of a FA in the binding site and not as previously assigned a network of water molecules. The ligand cis-vaccenic acid was easily modeled at full occupancy (Figure 1A). We observed clear electron density for two molecules of n-heptyl-β-D-glucopyranoside, binding on either side of the noncrystallographic 2-fold axis of symmetry, ∼12 Å from the FA head group, beside helix AF-2. This chemical was an essential additive for the first crystallization of PPAR-β/δ, by Xu et al., 1999Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Steinbach D.D. Lehman J.M. Wisely G.B. Willson T.M. et al.Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar. The additive molecules interact with each other and with each LBD (Figure 1B). Neither the FA nor the pyranoside ligands were defined in the previous work. A comparison of crystallographic statistics between the original and our analysis (Table 1) indicates that the structures are comparable. We note that the redetermined structure contains fewer residues and water molecules with an improvement in the Ramachandran plot mainly due to removal of poorly defined residues.Table 1Crystallographic StatisticsStructure2GWX (Old)2BAW (New)Wavelength (Å)1.54Unit cell dimensions0a (Å)39.77b (Å)94.22c (Å)96.70β (°)97.77Space groupP21Resolution range (Å)20.0–2.3Unique reflections26881RedundancyNRCompleteness (%)99.0 (71.2)<I / σ(I)>19.9 (6.6)Rsym (%)9 (NR)Wilson B (Å2)37.0Protein Residues/Atoms531/4201493/4026Residues in molecule A211–476211–241, 246–264, 275–475Residues in molecule B211–477211–238, 246–262, 275–477Water molecules185161C18:1 ligand−4Glucopyranoside−4R/Rfree (%)24.6/28.824.5/27.6Average B (Å2)Protein42.336.6C18:1−42.5Glucopyranoside−43.1Waters45.242.6RMSD bond lengths (Å)0.0080.009RMSD bond angles (°)1.501.23Cruickshank's DPI (Å)0.220.20Ramachandran plot (%)Residues in:Most favored region85.392.4Additionally allowed regions11.97.0Generally allowed regions2.60.2Disallowed0.20.4Values in parentheses pertain to the highest resolution shell (width ≈ 0.1 Å). Abbreviations: DPI, diffraction-component precision index (Cruickshank, 1999Cruickshank D.W.J. Acta Crystallogr. 1999; D55: 583-601Google Scholar); NR, not reported. Open table in a new tab Values in parentheses pertain to the highest resolution shell (width ≈ 0.1 Å). Abbreviations: DPI, diffraction-component precision index (Cruickshank, 1999Cruickshank D.W.J. Acta Crystallogr. 1999; D55: 583-601Google Scholar); NR, not reported. Our reevaluation of this structure serves to correct the published model, defining ligands previously overlooked. The binding site within the "apo structure" is actually occupied with FA likely derived from the expression system and that is why PPAR-β/δ displays the activated conformation. Importantly, our analysis suggests that binding data accumulated on the association of PPAR-β/δ LBD with ligands must be viewed with caution unless there is evidence that FA has been removed prior to analysis. Furthermore, in a biological context, the possibility exists that this nuclear hormone receptor is constitutively active, because it clearly binds endogenous FA ligands tightly. The new coordinates are deposited at the PDB (2BAW). Our study has been supported by BBSRC, LEO Pharma, ESRF, Daresbury laboratory, and the Wellcome Trust. We thank C. Bond for discussions.