In the discovery of new ligands from the lab in-house natural products library against RXRα and PPARγ, we construct a screening platform based on in-cell mammalian one hybrid assays. Among the natural products with the activities to activate either RXRα or PPARγ, magnolol unexpectedly shows its agonistic functions on both of these two nuclear receptors, with EC50 values of 10.4 µM and 17.7 µM, respectively (). Additionally, the magnolol-induced RXRα and PPARγ activations can be suppressed by the known RXRα and PPARγ antagonists HX531 and GW9662, respectively (), implying that magnolol takes its effects by targeting both of these two nuclear receptors. We further perform surface plasmon resonance (SPR) technology based experiments to detect the physical binding of magnolol to the purified RXRαLBD and PPARγLBD. As indicated in , magnolol dose-dependently binds to RXRαLBD and PPARγLBD with KD values of 45.7 µM and 1.67 µM, respectively.
As nuclear receptors, RXRα and PPARγ need to recruit their coactivators to initiate the transcription of target genes 
. Thus we further investigate whether magnolol can enhance these two nuclear receptors binding to the common coactivator steroid receptor coactivator-1 (SRC1) using SPR based technology. As indicated in , magnolol can increase RXRαLBD-SRC1 interactions in a dose-dependent manner. However, this natural product exhibits no effect on SRC1 recruitment to PPARγLBD (). Considering there are many other coactivators for PPARγ function 
, magnolol may probably take its effect by recruiting other coactivator instead of SRC1 for PPARγ involved transcription.
In activation of the downstream genes transcription, RXRα and PPARγ have to form RXRα:RXRα homodimer and RXRα:PPARγ heterodimer binding to their response elements. Thus we further evaluate the effects of magnolol on the activities of RXRα:RXRα homodimer and RXRα:PPARγ heterodimer using transactivation analyses on their response elements RXRE and PPRE. As indicated in , magnolol induces the transcription of PPRE in a dose-dependent manner. However, this compound exhibits no activity on RXRE transcription. Moreover, the magnolol-effect on PPRE transcription can be suppressed by both RXRα and PPARγ antagonists HX531 and GW9662, respectively (), which is in good accordance to our in-cell mammalian one hybrid assays (). It thus indicates that magnolol binding to both RXRα and PPARγ is required to activate PPRE transcription. Additionally, magnolol exhibits lower activities in their lower concentrations, compared to PPARγ agonist Rosiglitazone (). However, magnolol surprisingly shows equal activities to Rosiglitazone in their high concentrations, indicating magnolol is a PPARγ full agonist (). In conclusion, we identify magnolol from the natural product library functioning as a dual agonist of both RXRα and PPARγ, with the biased transcriptional activity on PPRE instead of RXRE.
Magnolol as a biased agonist on PPRE transcription.
As indicated in the previously reported crystal structures of RXRα ligand-binding domain complex with agonists, the essential activation function-2 (AF-2) motif in RXRα exhibits significant conformational changes. AF-2 motif overturns itself to cover the ligand-binding pocket upon agonist binding, thus exposing the surface for recruiting the coactivator SRC1 and initializing the transcription of target genes 
. The typical chemical structure of RXRα agonist consists of the acidic and hydrophobic moieties to adapt the L-shaped ligand-binding pocket of RXRα 
. Different from previously reported RXRα agonists, magnolol possesses two identical 5-allyl-2-hydroxyphenyl moieties. Thus we wonder how magnolol functions as an agonist of RXRα. To reveal the molecular basis for magnolol binding and activating RXRα, we determine the crystal structure of RXRαLBD-magnolol complex with SRC1 coactivator peptide. Magnolol-bound RXRαLBD exhibits a dimeric packing of RXRα. The electron density around magnolol is shown in . Magnolol binds into the hydrophobic ligand-binding pocket, and induces conserved conformational changes of AF-2 motif for SRC1 coactivator peptide recruitment. Magnolol is found to adapt itself to an L-shaped conformation, with two 5-allyl-2-hydroxyphenyl moieties occupying each side of the L-shaped pocket, respectively. The typical RXRα agonists always form a hydrogen bond with Arg316 in the C-terminus of helix 5 
. However, magnolol uses one hydroxyl group to form a hydrogen bond with Asn306 in the N-terminus of helix 5 (). Such an interaction induces an overturning of Asn306, compared with the known agonist 9-cis
-retinoic acid-bound RXRαLBD structure (). Moreover, helix 3 is observed to bend towards the ligand-binding pocket from its position in apo RXRαLBD structure, which is consistent with the known agonist-bound RXRαLBD structures 
. Therefore, from our determined crystal structure of RXRαLBD-magnolol-SRC1, the agonist magnolol employs a distinct binding mode for RXRα activation by interacting with Asn306 in the N-terminus of helix 5, instead of Arg316 in the C-terminus of helix 5. And magnolol adapts its two 5-allyl-2-hydroxyphenyl moieties occupying the hydrophobic and acidic sides of the pocket, respectively.
Crystal structures of RXRαLBD-magnolol-SRC1 and PPARγLBD-magnolol.
Different from RXRα with the L-shaped ligand-binding pocket, PPARγ uses a much larger Y-shaped pocket for ligand-binding 
. And PPARγ ligand-binding pocket can be divided into two sub-pockets, AF-2 sub-pocket and β-sheet sub-pocket 
. PPARγ agonists are categorized as full and partial agonists, depending on their activities in the cell-based reporter assays 
. It is suggested that PPARγ partial agonists bind only β-sheet sub-pocket, while full agonists always occupy both AF-2 and β-sheet sub-pockets to activate PPARγ 
. Magnolol is determined to be a full agonist of PPARγ in the current work (). Thus we wonder how magnolol binds such a Y-shaped pocket for PPARγ activation. In our determined crystal structure of PPARγLBD-magnolol, the electron density map around magnolol is shown in . Interestingly, two magnolol molecules are found in PPARγ ligand-binding pocket, one in AF-2 sub-pocket and the other in β-sheet sub-pocket. The hydroxyl group of magnolol in AF-2 sub-pocket forms a hydrogen bond with Ser289 in helix 3, as well as water-mediated hydrogen bonds with Tyr473 in AF-2 motif (). Direct interactions between agonist and AF-2 motif are believed to play a crucial role in the conformational changes of PPARγ AF-2 motif, and surface formation for coactivator recruitment 
. On the other side, the hydroxyl group of magnolol in β-sheet sub-pocket interacts with Ser342 in β-sheet with a hydrogen bond (). Moreover, there is also a water-mediated hydrogen bond with magnolol in β-sheet sub-pocket to further stabilize the ligand binding (). Our findings have thus revealed an unexpected binding mode of magnolol on PPARγ, with two identical chemical compounds binding two different sub-pockets, which probably lead for new PPARγ agonists design.
To evaluate the degree of cooperativity of the two magnolol molecules binding to PPARγ, Hill coefficient is determined. The value of approximately 2 indicates that magnolol binding is positively cooperative, and both the binding sites can bind magnolol simultaneously. Thus two magnolol molecules cooperatively induce PPARγ activation by interacting with both AF-2 motif and β-sheet, respectively. Furthermore, the fact that two magnolol molecules cooperatively bind to PPARγ also explains the reason why magnolol exhibits lower activities on PPRE transcription, compared to PPARγ agonist Rosiglitazone (). Although magnolol and Rosiglitazone are both PPARγ full agonists, their transactivation curves indicate their different mechanisms (). Only one molecule of Rosiglitazone is necessary for PPARγ activation, while two magnolol molecules are required to bind PPARγ. Considering that the magnolol-effect on PPRE transcription can also be suppressed by RXRα antagonist HX531, and HX531 can inhibit RXRα agonist 9cRA activity on PPRE, it thus suggests that magnolol binding to RXRα is also necessary for PPRE transcription. Therefore, totally three magnolol molecules are required for PPRE transcription, with one molecule binding to RXRα and two molecules binding to PPARγ.
Magnolol was once characterized as a PPARγ agonist with the computer aided modelling 
. However, our co-crystal structure of PPARγLBD-magnolol reveals a distinct ligand binding mode. As indicated in , magnolol in AF-2 sub-pocket is found to form not only a hydrogen bond with Ser289 in helix 3, but also water-mediated hydrogen bonds with Tyr473 in AF-2 motif. On the other side, in β-sheet sub-pocket of PPARγ, magnolol interacts with Ser342 in β-sheet (), instead of Gly284 that was determined by the computer aided modelling. Moreover, we also find a water-mediated hydrogen bond with magnolol in β-sheet sub-pocket to further stabilize the ligand binding (). Considering that the water-mediated interactions within PPARγLBD-magnolol is still delicate to be determined by the computer based modelling, our co-crystal structure is expected to supply further insights into the future computer based modelling.
Honokiol, an analogue of magnolol, shares some certain biological properties with magnolol 
. And honokiol was reported to have anti-angiogenic, anti-inflammatory and antitumor functions, but the mechanisms of honokiol actions are still elusive. Here we find that magnolol targets both RXRαLBD and PPARγLBD, thus how honokiol interacts with these two nuclear receptors will be of potentially important and interesting. Moreover, knowledge of mechanisms of magnolol and honokiol actions may assist novel synthetic analogues development in the future.
From the RXRαLBD-magnolol and PPARγLBD-magnolol structures, it is suggested that the hydroxyl groups of magnolol play essential roles in the receptor-ligand interactions. In RXRαLBD-magnolol structure, the hydroxyl group of magnolol contacts with Asn306 in helix 5 of RXRα. While, in PPARγLBD-magnolol structure, the hydroxyl groups from the two bound ligands interact with Ser342 in β-sheet, Tyr473 in AF-2 motif, and Ser289 in helix 3 of PPARγ, respectively. Additionally, magnolol adopts surprising binding modes on these two nuclear receptors. Although magnolol is big enough to accommodate mostly the L-shaped RXRα ligand-binding pocket, two magnolol molecules have to cooperatively occupy the much larger Y-shaped PPARγ ligand-binding pocket. Furthermore, the single bond connecting the two 5-allyl-2-hydroxyphenyl moieties of magnolol endows this chemical compound flexibility to fit the different pocket sizes of RXRα and PPARγ. As shown in , magnolol molecules exhibit three different conformations when it binds to RXRα and PPARγ. show the key secondary structures of RXRα and PPARγ, with which magnolol makes direct interactions. Our findings are in good accordance with that the homo-/heterodimeric interface and coactivator binding surface of RXRα and PPARγ are critical for both of these two nuclear receptors activation. And all of these secondary structures of RXRα and PPARγ are conserved in the agonist binding and interactions. Considering the large differences between RXRα L-shaped pocket and PPARγ Y-shaped pocket, future dual agonist design may focus on PPARγ sub-pockets, since each PPARγ sub-pocket has a similar size to the whole pocket of RXRα. The agonist which can accommodate to RXRα ligand-binding pocket and the two PPARγ sub-pockets with preferred activities will probably have potentials to activate both of these two nuclear receptors.
Key interactions for magnolol function on RXRα and PPARγ.