Title: Combined Simulation and Mutagenesis Analyses Reveal the Involvement of Key Residues for Peroxisome Proliferator-activated Receptorα Helix 12 Dynamic Behavior
Abstract: The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor α (PPARα) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARα helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARα helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARα transcription activation potential. Finally, we disclose important functions of residues in PPARα AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARα constitutive activity, without changing PPARα ligand-dependent activation potential. The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor α (PPARα) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARα helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARα helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARα transcription activation potential. Finally, we disclose important functions of residues in PPARα AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARα constitutive activity, without changing PPARα ligand-dependent activation potential. The three peroxisome proliferator-activated receptor isotypes (PPARs) 5The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; LBD, ligand binding domain; AF-1, -2, activation functions 1 and 2; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; MD, molecular dynamics; wt, wild type; r.m.s.d., root mean square deviation; PPRE, peroxisome proliferator response element. 5The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; LBD, ligand binding domain; AF-1, -2, activation functions 1 and 2; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; MD, molecular dynamics; wt, wild type; r.m.s.d., root mean square deviation; PPRE, peroxisome proliferator response element. α, β/δ, and γ (NR1C1, NR1C2, and NR1C3, respectively (1Committee Nuclear Receptors Nomenclature Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (934) Google Scholar)) form a distinct subfamily of nuclear hormone receptors (2Desvergne B. 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Motif I ends four residues upstream of the C-terminal end of the protein, whereas motif II corresponds to the last six residues of the receptor. Because PPARs are therapeutic targets, it is important to understand the molecular basis of helix 12 constitutive and ligand-dependent dynamics, since this may provide valuable information with regard to drug design. In this report, we have analyzed the role of these C-terminal residues in PPARα activity using combined experimental mutant analyses and computational simulations. We have identified important residues for PPARα, which determine helix 12 regulation in its constitutive and ligand-dependent transcriptional activity. Based on these results, a detailed mechanistic description of the ligand-dependent role of helix 12 is proposed. Xenopus laevis PPARα mutants were obtained as described previously (29Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4557) Google Scholar). HeLa cells cultured in 10% delipidated fetal calf serum (Invitrogen) were transfected with pSG5 expression vectors containing full-length wild-type or mutant PPARα, and either CAT (ACO-A pBL-CAT8+ (30Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1197) Google Scholar)) or Luciferase (3xACOPPRE-TK-Luc) reporter plasmids, as indicated in the figure legends. PPARα agonist Wy-14,643 (Chem Syn Laboratories) was added 6 and 24 h after transfection. 48 h after transfection (Lipofectamine 2000, Invitrogen), cell extracts were prepared by freeze-thawing and were assayed for CAT or luciferase activity (Promega, Madison, WI). The GST-p3002–516 (31Gelman L. Zhou G. Fajas L. Raspe E. Fruchart J.C. Auwerx J. J. Biol. Chem. 1999; 274: 7681-7688Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) fusion protein was expressed in Escherichia coli and purified on a glutathione affinity matrix (Amersham Biosciences). Full-length PPARs were produced with reticulocyte lysates (TnT T7 quick translation/transcription system) and labeled with [35S]methionine. The GST-p300 fusion protein or the GST protein alone (3 μg each) were then incubated with 15 μl of programmed reticulocyte lysates in 500 μl of binding buffer (Tris-HCl, pH 7.4 25 mm, EDTA 1 mm, NaCl 100 mm, Triton X-100 0.1%, phenylmethylsulfonyl fluoride 0.2 mm, protease inhibitor mixture (Roche Applied Science)) supplemented with 0.5% dry milk, during 4 h at 4°C, in the presence or absence of the PPARα ligand Wy-14,643 at 100 μm. Beads were washed three times with binding buffer, and samples were boiled with 40 μl of 2× SDS-PAGE buffer (12.5 mm Tris-HCl, 20% glycerol, 0.002% Bromphenol Blue, 5% β-mercaptoethanol), separated on a 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and exposed to a phosphorimager (PhosphorImager, Storm 840, Amersham Biosciences). Deriving the Unliganded PPARα Model—The homology model of the xPPARα ligand binding domain (SwissProt accession number P37232) was built using MODELLER v6.2 (32Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10456) Google Scholar) based on the crystal structure of the human PPARα (Protein Data Bank (PDB) (33Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (26989) Google Scholar, 34Berman H.M. Battistuz T. Bhat T.N. Bluhm W.F. Bourne P.E. Burkhardt K. Feng Z. Gilliland G.L. Iype L. Jain S. Fagan P. Marvin J. Padilla D. Ravichandran V. Schneider B. Thanki N. Weissig H. Westbrook J.D. Zardecki C. Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 899-907Crossref PubMed Scopus (1840) Google Scholar) code 1K7L (21Xu H.E. Lambert M.H. Montana V.G. Plunket K.D. Moore L.B. Collins J.L. Oplinger J.A. Kliewer S.A. Gampe Jr., R.T. McKee D.D. Moore J.T. Willson T.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13919-13924Crossref PubMed Scopus (458) Google Scholar)). In this structure, helix 12 is in the closed conformation. ClustalW (35Thompson S.K. Murthy K.H. Zhao B. Winborne E. Green D.W. Fisher S.M. DesJarlais R.L. Tomaszek Jr., T.A. Meek T.D. Gleason J.G. Abdelmeguid S.S. J. Med. Chem. 1994; 37: 3100-3107Crossref PubMed Scopus (71) Google Scholar) was used to perform a pairwise sequence alignment of the X. laevis and human sequences. The sequence identity is 90%. Default parameters of the homology modeling routine were used. The energy of the model was then minimized using the CHARMM program (36Brooks B.R. Bruccoleri R. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (13881) Google Scholar) and the CHARMM19 force field (37Neria E. Fischer S. Karplus M. J. Chem. Phys. 1996; 105: 1902-1921Crossref Scopus (1093) Google Scholar, 38Reiher W.E. II I Thesis.Theoretical Studies of Hydrogen Bonding. Dept. of Chemistry, Harvard University, Cambridge, MA1985Google Scholar), with a dielectric constant of 1 and a 20 Å cutoff. The minimization consisted of 30 steps of steepest descent followed by 30 steps of adopted basis Newton-Raphson. The positions of the Cα atoms were constrained using a mass-weighted harmonic force constant of 10 kcal/(mol Å2). Docking of Wy-14,643—Missing parameters for the ligand, for use in conjunction with the CHARMM22 (39MacKerell A.D. Bashford D. Bellott M. Dunbrack R.L. Evanseck J.D. Field M.J. Fischer S. Gao J. Guo H. Ha S. McCarthy D.J. Kuchnir L. Kuczera K. Lau F.T.K. Mattos C. Michnick S. Ngo T. Nguyen D.T. Prodhom B. Reiher W.E. Roux B. Schlenkrich M. Smith J.C. Stote R. Straub J. Watanabe M. Kuczera J.W. Yin D. Karplus M. J. Phys. Chem. B. 1998; 102: 3586-3616Crossref PubMed Scopus (11621) Google Scholar) force field, were derived from the Merck Molecular Force Field (MMFF (40Halgren T.H. J. Comput. Chem. 1996; 17: 616-641Crossref Scopus (648) Google Scholar, 41Halgren T.H. J. Comput. Chem. 1996; 17: 587-615Google Scholar, 42Halgren T.H. J. Comput. Chem. 1996; 17: 553-586Crossref Scopus (733) Google Scholar, 43Halgren T.H. J. Comput. Chem. 1996; 17: 520-552Crossref Scopus (986) Google Scholar, 44Halgren T.H. J. Comput. Chem. 1996; 17: 490-519Crossref Scopus (4242) Google Scholar)). Based on the PPARα model mentioned above, a model of the PPARα/Wy-14,643 complex was built using the EADock evolutionary algorithm, taking account of the solvent effect. Details of the calculations are presented separately (45Grosdidier A. Zoete V. Michielin O. Proteins. 2007; (in press)Google Scholar). Residues of the binding site were flexible during the docking to account for the inherent inaccuracy of coordinates in the PPARα model, and for the induced fit of the protein in the presence of the ligand. These include residues 247, 253, 257, 278–279, 281–283, 285–286, 320, 323–324, 327, 336, 338, 345, 360–361, 446, 450, and 470. The final conformations with the lowest energy were further minimized by 100 steps of steepest descent using the GB-MV2 (46Lee M.S. Salsbury Jr., F.R. Brooks III, C.L. J. Chem. Phys. 2002; 116: 10606-10614Crossref Scopus (398) Google Scholar, 47Lee M.S. Feig M. Salsbury Jr., F.R. Brooks 3rd., C.L. J. Comput. Chem. 2003; 24: 1348-1356Crossref PubMed Scopus (417) Google Scholar) generalized Born model. The lowest energy conformation was used for the following molecular dynamics (MD) simulations. MD Simulations—All simulations were performed using the CHARMM program (version c31b1) and the CHARMM22 force field. The starting structure of the R471L/D472N PPARα mutant was obtained from the structure of the wild-type protein by replacing the two native side chains by the mutated ones. MD simulations were performed on four systems, corresponding to the wild-type or mutated PPARα, in the presence (liganded) or in absence (unliganded) of the Wy-14,643 ligand. Each isolated system (no co-activator) was minimized using 250 steps of steepest descent minimization using the GBSW implicit solvent (48Im W. Lee M.S. Brooks 3rd., C.L. J. Comput. Chem. 2003; 24: 1691-1702Crossref PubMed Scopus (549) Google Scholar) to remove sterical clashes in the structural model. The protein (or complex) was solvated in a cubic box of 80.7 Å3 of TIP3P (49Jorgensen W.L. Chandrasekhar J. Madura J.D. Impey R.W. Klein M.L. J. Chem. Phys. 1983; 79: 926-935Crossref Scopus (29504) Google Scholar) water molecules that were previously equilibrated at 300 K and 1 atm of pressure. The solvent was equilibrated at 300 K during 20 ps in the presence of the fixed protein. The entire system was then equilibrated at 300 K during 150 ps, in the isothermal isobaric ensemble (NPT) to adjust the solvent density at 1 atm. Finally, the production MD simulation was conducted during 1 ns in the canonical (isovolume isothermal, NVT) ensemble. The MD simulations were performed with periodic boundary conditions. The Verlet leapfrog integrator was used for time propagation with a time step of 0.001 ps. A 12-Å cutoff was applied. Calculation of Side-chain Contributions to the Conformational Stability—To estimate the role of the residue side chains on the protein stability, we used the approach developed by V. Zoete and M. Meuwly (52Zoete V. Meuwly M. J. Comput. Chem. 2006; 27: 1843-1857Crossref PubMed Scopus (43) Google Scholar). The method is based on the notion that the binding free energy (ΔGstab) corresponding to the alchemical complexation of a given side chain (considered as a "pseudo-ligand") into the rest of the protein (considered as a "pseudo-receptor") reflects the importance of this side chain to the thermodynamic stability of the protein (52Zoete V. Meuwly M. J. Comput. Chem. 2006; 27: 1843-1857Crossref PubMed Scopus (43) Google Scholar). The binding free energy was estimated according to the MMGBSA approach, ⟨ΔGbind⟩=⟨EvdW⟩+⟨Eelec⟩+⟨ΔEintra⟩+⟨ΔGelec,desolv⟩+⟨ΔGnp,desolv⟩-T⟨ΔS⟩(Eq. 1) where the brackets, 〈 and 〉, indicate an average of these energy terms over 250 frames regularly extracted from the 1-ns MD simulation trajectory of the complex described above (one every 4 ps). Terms relative to a given isolated partner were also calculated using frames extracted from the MD simulation of the complex, after removing the other partner. This single trajectory method was found to lead to important cancellation of errors (53Zoete V. Meuwly M. Karplus M. Proteins. 2005; 61: 79-93Crossref PubMed Scopus (190) Google Scholar). EvdW and Eelec are the van der Waals and electrostatic pseudo-ligand:pseudo-receptor interaction energies, respectively. ΔEintra is the variation of the internal energy of both partners upon complexation. Because we use the single trajectory method, we have in fact ΔEintra = 0. ΔGelec,desolv and ΔGnp,desolv are the electrostatic and non-polar desolvation energies, respectively. ΔGelec,desolv is calculated according to the GB-MV2 generalized Born model. ΔGnp,desolv is assumed to be proportional to the solvent-accessible surface area that is buried upon complexation (54Amidon G.L. Yalkowsky S.H. Anik S.T. Valvani S.C. J. Phys. Chem. 1975; 79: 2239-2246Crossref Scopus (132) Google Scholar, 55Hermann R.B. J. Phys. 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Integrity of Motifs I and II of PPARα Helix 12 Is Required for the Full Transcriptional Activity of the Receptor—Using X. laevis PPARα (PPARα) as a model (30Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1197) Google Scholar), we studied whether motifs I and II of helix 12 are both necessary for the function of the PPARs. Mutants were generated in which 1, 2, 4, or 12 C-terminal residues were deleted (Δ1, Δ2, Δ4, and Δ12, respectively). Of these, Δ1, Δ2, and Δ4 affected motif II residues, whereas Δ 12 affected the entire helix 12. The mutants were tested for their ability to activate a PPRE-driven reporter construct using transient transfections in HeLa cells in the presence or absence of Wy-14,643, a selective PPARα ligand (59Keller H. Devchand P.R. Perroud M. Wahli W. Biol. Chem. 1997; 378: 651-655Crossref PubMed Scopus (89) Google Scholar) (Fig. 2A). The mutations did not affect the ectopic expression level of these proteins relative to wt PPARα (data not shown). As expected, deletion of both motif I and II (Δ 12) resulted in total loss of transcriptional activity (Fig. 2A). Interestingly, total loss of activity was also observed with mutant Δ4, which lacks motif II, suggesting that motif I on its own is unable to sustain transactivation. The integrity of motif II as a prerequisite for full transactivation was confirmed with mutants Δ1 and Δ2, which exhibited decreased transcriptional activity. These first experiments also confirmed that PPARα has a relatively high constitutive activity in the absence of any exogenous ligand (10Juge-Aubry C.E. Hammar E. Siegrist-Kaiser C. Pernin A. Takeshita A. Chin W.W. Burger A.G. Meier C.A. J. Biol. Chem. 1999; 274: 10505-10510Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 59Keller H. Devchand P.R. Perroud M. Wahli W. Biol. 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To further investigate which residues are responsible for the transactivation properties of the receptor, point mutations on helix 12 residues were generated. The acidic residue centrally located within the AF-2 core motif of estrogen receptor, thyroid hormone receptor, and retinoic acid receptor, was shown to be essential for the ligand-dependent activation function of these receptors (61Barettino D. Vivanco Ruiz M.M. Stunnenberg H.G. EMBO J. 1994; 13: 3039-3049Crossref PubMed Scopus (290) Google Scholar, 62Tone Y. Collingwood T.N. Adams M. Chatterjee V.K. J. Biol. Chem. 1994; 269: 31157-31161Abstract Full Text PDF PubMed Google Scholar, 63Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (315) Google Scholar, 64Feng X. Peng Z.H. Di W. Li X.Y. Rochette-Egly C. Chambon P. Voorhees J.J. Xiao J.H. Genes Dev. 1997; 11: 59-71Crossref PubMed Scopus (59) Google Scholar, 65Tzukerman M.T. Esty A. Santiso-Mere D. Danielian P. Parker M.G. Stein R.B. Pike J.W. McDonnell D.P. Mol. Endocrinol. 1994; 8: 21-30Crossref PubMed Scopus (608) Google Scholar, 66Cavailles V. Dauvois S. L'Horset F. Lopez G. Hoare S. Kushner P.J. Parker M.G. EMBO J. 1995; 14: 3741-3751Crossref PubMed Scopus (671) Google Scholar). Therefore, we first targeted Glu at position 468 (motif I) and Asp-472 (motif II), which were replaced by Gln and Asn, respectively (E468Q and D472N). The E468Q mutant was totally inactive in the transactivation assay, in the presence or absence of ligand (Fig. 2B). The D472N mutation had a less dramatic effect, because the receptor retained 35% of the constitutive and 60% of the induced activity of the wt PPARα. One feature distinguishing motif II from motif I in all known PPARs is the presence of a positively charged residue, Arg or Lys, preceding the centrally located Asp residue (Fig. 1). Replacement of Arg-471 with Leu (R471L) resulted in a disproportionate decrease of the constitutive relative to the induced activity (Fig. 2C). The simultaneous substitution of residues Arg-471 and Asp-472 to Leu and Asn, respectively (R471L/D472N), resulted in further decrease in the ligand-dependent but in no cumulative decrease in the constitutive activity, compared with the single mutant D472N (Fig. 2C). The possibility that post-translational modifications affect the function of the AF-2 domain was examined by substituting Tyr-470, a potential target of phosphorylation by various kinases, with Phe (Y470F, Fig. 2B). This mutation resulted in a receptor retaining 80% of wt PPARα activity, indicating that phosphorylation of this residue, if it takes place, is not crucial for PPARα activity. Together, the above results suggest that the PPARα AF-2 function depends on the integrity of both motifs I and II and confirm the crucial role of the acidic residues Glu-468 (motif I) and Asp-472 (motif II). Point mutants were then designed according to the data obtained after free energy simulations of the wt PPARα LBD. As presented in detail later in this report, an in silico approach was used to determine the contribution of each residue in the LBD to the protein structural stability. It showed that in helix 12, Leu-466, Met-473, and Tyr-474 are associated with large negative ΔG value and that they most probably strongly stabilize the active conformation of the helix (Table 1). Each of these residues was mutated into the neutral residue Ala (L466A, M473A, and Y474A). A double mutation of the C-terminal residues Met-473 and Tyr-474 was also generated (M473A/Y474A), and the activity of these mutants was tested in the transactivation assay (Fig. 2D). Mutation of Leu-466 almost totally suppressed the activity of the receptor. M473A and Y474A retained 30 and 80% of wt activity, respectively. Interestingly, M473A/Y474A lost constitutive activity but retained 30% of the ligand-inducible activity, like the Δ2 deletion mutant. Transactivation assays thus confirmed the in silico prediction according to which Leu-466, Met-473, and Tyr-474 are important residues for the activity of the receptor. Taken together, all our mutagenesis results argue for a r