Molecular Dynamics Simulation of the Ligand Binding Domain of

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Molecular Dynamics Simulation of the Ligand Binding Domain of Farnesoid X Receptor. Insights into Helix-12 Stability and Coactivator Peptide Stabilization in Response to Agonist Binding Gabriele Costantino,*,§ Antonio Entrena-Guadix,§,‡ Antonio Macchiarulo,§ Antimo Gioiello,§ and Roberto Pellicciari§ Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di Perugia, Via del Liceo 1, 06123 Perugia, Italy, and Departamento de Quı´mica Farmace´ utica y Orga´ nica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n, 18071 Granada, Spain Received October 13, 2004

The dynamic changes which take place in the ligand binding domain (LBD) of farneosid X receptor (FXR) in response to agonist binding and in the presence of coactivator peptides were studied with nanosecond time-scale molecular dynamics. Four different systems were analyzed, including the holo-LBD complexed with 6ECDCA, the holo-LBD in the presence of two coactivator peptides, and two artificial apo forms, with and without coactivator peptides. Our results revealed a detailed picture of the differential micro- and macromodifications occurring in the LBD in the presence or not of the agonist molecule and the coactivator peptides. In the apo simulation a major conformational change took place in the crucial helix 12, while the holo-LBD was globally stabilized by the ligand. When the coactivator peptides were included in the simulation, a clear agonist-induced stabilization was observed for the canonical peptide. Interestingly, the second peptide was released from the holo-LBD while it was kept bound in the apo simulation. The present results provide a molecular basis for the understanding the role played by the bile acid agonist in receptor stabilization and enhanced cofactor recruitments. Introduction Nuclear receptors (NRs) constitute one of the largest families of transcription factors which regulate target gene expression or repression.1 The transcriptional activity of NRs is regulated by a complex network of interaction which includes recognition by the endogenous ligand, homo- or heterodimerization, cross-talk between the activation functions and the DNA binding domain, and release of corepressor and recruitment of coactivator peptides. The ligand-dependent transcriptional activity of NRs is confined in the carboxy terminal domain (CTD), which contains the binding sites for the ligand and for the coactivator (or corepressor) peptides. In recent years, a considerable number of crystallographic structures of the ligand binding domain (LBD) of several NRs have appeared in the literature and been made available through the Brookhaven Protein Database (PDB). Inspection of these structures has revealed a highly conserved architecture constituted by an antiparallel sandwich of 11 to 13 R-helices.2 The binding of agonist molecules causes a conformational change of the activation function-2 (AF-2), usually helix 12 (H12), thus permitting an efficient recruitment of the coactivator peptide which bears a conserved LxxLL sequence.3 While this ligand-induced conformational change is considered a conserved and essential feature of all the NRs, the molecular details of the pathways leading from ligand binding to target gene expression (or repression) are still to be fully clarified. For instance, a promiscuity * Corresponding author. E-mail: [email protected], Phone: +39 075 585 5160, Fax: +39 075 585 5165. § Universita ` di Perugia. ‡ Universidad de Granada.

of target genes is usually transcripted by the same receptor when activated by structurally diverse ligands. This observation leads to the point that different chemotypes are apparently able to recruit different coactivators or to (de)stabilize to different extent various receptor-ligand-coactivator complexes, thus leading to the differential interaction with different gene promoters. Of particular interest in this context is the FXR receptor, first identified five years ago as the physiological sensor for endogenous bile acids (BA).4-6 FXR responds to physiological concentration of the naturally occurring chenodeoxycholic acid (CDCA, Chart 1) 1 and is potently activated by the synthetic analogue 6-ethylchenodeoxycholic acid (6ECDCA) 27 and by nonsteroid ligands such as GW4611 (3)8 and fexaramine (4).9 When activated by agonists, FXR represses the expression of target genes, such as CYP7A and CYP8B,10 through the increase in the level of the nuclear receptor SHP,11,12 and promotes the gene expression of the bile acid transporters I-BABP, BSEP, and MAOT.13 The intimate role of FXR in the transcriptional control of genes regulating cholesterol and BA biosynthesis and disposal, makes it a particularly attractive target for the discovery of drugs for the treatment of cholestasis7 or hyperlipidemia.14 Notably, different chemotypes (bile acid-like, GW-like, fexaramine-like) promote and induce different patterns of gene expression9,15-16 thus raising the point of the identification of the molecular determinants linking ligand binding, coactivator recruitment, and the target gene expression. The disclosure of crystallographic structures of the LBD of FXR complexed with two bile

10.1021/jm049182o CCC: $30.25 © 2005 American Chemical Society Published on Web 04/09/2005

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Chart 1

Figure 1. General view of the Chain B in the crystal structure of the LBD of the FXR receptor (1OSV).17 Red: AF2; green: Coactivator 1; purple: coactivator 2.

acid agonists (6ECDCA and 3-deoxy-CDCA)17 and with the nonsteroid ligand fexaramine9 can help in answering some of the structural and mechanistic questions. The inspection of the complexes between the LBD of ratFXR17 and the bile acid analogues revealed the canonical structural arrangement of NRs, with three peculiar features: (i) the bile acid ligand has the steroid backbone flipped head-to-tail with respect to the binding of other steroid hormone to their respective cognate receptors. This peculiar feature was correctly anticipated by us in a homology-based molecular modeling study;18 (ii) the LBD of FXR was cocrystallized in the presence of a synthetic peptide corresponding to the NID-3 region of GRIP-1 coactivator; one monomer of the thus crystallographically determined dimer showed two bound LxxLL peptides: one in the ‘classical’ coactivator cleft, the other one disposed along helix-3 in an antiparallel fashion. Whether this fact has biological significance or it is due to crystallographic artifacts is still unknown, but the presence of two coactivator peptides is unprecedented in the NR superfamily; (iii) the helix 12, or AF2, is frozen by the bile acid agonist in the ‘classical’ active conformation. The stabilization of the active conformation is given by an unusual trigger constituted by a tyrosine-histidine-tryptophan triad, apparently stabilized by hydrophobic interaction with the ring A of the steroid backbone. The salient features of the LBD of rFXR complexed with 6ECDCA are depicted in Figure 1. Central to the understanding of the molecular role played by individual agonists on coactivator recruitment and target gene expression is the ability of predicting the overall conformational changes and the overall stability of the receptor upon ligand binding. In this respect, crystallographically determined structures provide essential but not conclusive information, as being representative of ‘extreme’ states of the configurational space of the receptor-ligand-coactivator complex. Given these considerations, we thought that a computational study based on nanosecond time-scale molecular dynamics simulation constitutes an appropriate approach for analyzing the dynamic response of the receptor upon ligand binding and may provide the theoretical grounds for further experimental validation. More in details, we sought to carry out molecular dynamics (MD) simulations which will specifically ad-

dress the following issues related to the mutual interaction between LBD, ligand and coactivator peptide(s): i. The stability of the ‘active’ conformation of LBD when the ligand is removed from the binding pocket, with a special emphasis given to the behavior of H12. ii. The influence of the bound peptide(s) in the stabilization (or possible destabilization) of the ‘active’ conformation of LBD. iii. The identification of the features responsible for the stabilization of the LBD’s conformation during the simulation. iv. The possible role of the second, noncanonical, peptide bound to LBD. These issues will be studied on the basis of four independent simulations. In the first set of simulations, the holo-LBD of FXR, complexed with the potent agonist 6ECDCA 2, was studied in the presence or not of the two coactivator peptides (simulation A and simulation B, respectively. See Table 1 for details). These simulations are designed to shed light on the role of the bile acid agonist in stabilizing the ‘active’ receptor conformation and to decipher the potential molecular cross-talk between ligand and peptides. In the second set of simulations, the apo-LBD of FXR was studied in the presence or in the absence of the coactivator peptides (simulation C and D, respectively). It should be mentioned that up to now no crystal structures of the apoLBD of FXR are available; thus, we started the simulations from the ‘active’ conformation of the LBD after having removed the ligand from the binding pocket. The aim of simulation C and D is to investigate the stability of the active conformation and the potential stabilization of the peptides in the absence of the ligand. It should be noticed that systems A and B represent experimentally observed states of the receptor, while systems C and D represent theoretically possible but not yet observed receptor configurations. Material and Methods The starting structure for the simulation was taken from the crystal structure of 6ECDCA complex (pdb code: 1OSV).17 Since one of our main purposes was the study of the potential role of the second coactivator peptide, chain B of 1OSV, which contains two bound peptides, was chosen for the analyses. Thus, chain B of the complex, the corresponding 6ECDCA molecule, both coactivator chains, and all the water molecules (74 molecules) surrounding chain B were extracted from the crystal structure and used when necessary for the construction of the initial geometry in each simulation.

Molecular Dynamics of FXR

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Table 1. Summary of the Molecular Dynamics Simulations simulation code

system composition

number of atoms

length of simulation

A

LBD of FXR; two coactivator peptides (coact1, coact2); agonist (6ECDCA), water

17.362

B

LBD of FXR; agonist (6ECDCA); water

17.410

C

LBD of FXR; two coactivator peptides (coact1, coact2); water

17.316

D

LBD of FXR; water

17.373

6 ps - heating time 200 ps - equilibration 3000 ps - production time 6 ps - heating time 200 ps - equilibration 3000 ps - production time 6 ps - heating time 200 ps - equilibration 3000 ps - production time 6 ps - heating time 200 ps - equilibration 3000 ps - production time

Hydrogen atoms were added using InsightII,19 and the Soak utility was used to add a sphere of TIP3P water molecules with a 35 Å radius. Atomic charges and potentials were fixed using CHARMM force-field, and both PDB and PSF files were written in X-PLOR format in order to export the structure to NAMD20 for the molecular dynamics simulations. After several initial minimizations carried out with the aim of relaxing nonbonding interactions in the system, a molecular dynamic equilibration of the solvent (100 ps) was performed by keeping frozen the heavy atoms of the protein and of the ligand, with the aim of allowing the water to move freely around and/or inside the protein. The last frame from this dynamic simulation was chosen as starting point for two new minimization processes, one of them with the CR atoms of the protein fixed and the other one with the system fully unrestricted. Finally, the molecular dynamics simulations were tackled in two stages. In the first one, the system was heated from 0 to 300 K, increasing the temperature 25 K for every 500 fs (total time: 6 ps), and, after that, equilibrated for 200 ps. The final coordinates and velocities of this stage were used to initiate a simulation of 3 ns. In all the simulations, spherical harmonic boundary conditions were used. For this purpose, a combination of two potentials centered on the mass center of the system was defined. For the first potential, a force constant of 100 kcal/ mol Å2 and a radius of 36 Å was defined, while for the second potential, the values of the force constant and of the radius were of 200 kcal/mol Å2 and 41 Å, respectively. During all the simulations, the BOUNDARY energy was less than 1 kcal/ mol, thus indicating that only a small number of water molecules arrived to the sphere potential and were pushed again into the sphere. During the production phase, temperature was kept constant by rescaling the temperature of the system every 500 fs. A time step of 1 fs was used, and no constraints such as the SHAKE protocol were employed. A snapshot of the system was written every 2 ps.

Results 1. Stability of the Simulations. The two systems A and B, where the bile acid agonist is bound to the LBD, were very stable during the 3 ns simulations (Table 2). The root-mean-square deviation (rmsd) calculated over the backbone atoms between the crystal structure and the average molecular dynamics structure were 1.61 ( 0.23 Å and 1.54 ( 0.15 Å, respectively. The radius of gyration of the two MD structures is likewise very similar to that of the crystal structure (Table 2). When systems C and D were examined, a major difference became apparent. While system C, in which the two coactivator peptides were kept bound to the LBD but the ligand was removed, had a rmsd as low as 1.57 ( 0.18 Å from the crystal structure and a radius of gyration comparable to that of the experimentally determined structure (see Table 2), the apo system D

Table 2. Structural Properties of Average MD Structures for Systems A-D crystal structure (experimental values)a

average MD structuresb

simulation

radius of gyration (Å)

radius of gyration (Å)

rmsd (Å)

A (ECDc)

18.22 (LBD and coact.s) 17.99 (LBD only) 17.99 (LBD only) 18.22 (LBD and coact.s) 17.99 (LBD only) 17.99 (LBD only)

18.22 ( 0.07 17.91 ( 0.08 17.95 ( 0.07 18.28 ( 0.07 18.03 ( 0.07 18.10 ( 0.07

1.61 ( 0.23 1.53 ( 0.21 1.54 ( 0.15 1.57 ( 0.18 1.59 ( 0.19 1.92 ( 0.61

B (ECD) C (FXRc) D (FXR)

a Value of the radius of gyration calculated over the crystal structure (pdb code: 1osv). b Values of the radius of gyration and rmsd from the crystal structure calculated for the four average MD structures. Standard deviations are given.

Figure 2. Variation of the radius of gyration for system D during the 3 ns trajectory. At t ) 1.3 ns the radius of gyration starts growing, indicating an expansion of the receptor due to the movement of AF2.

behaved differently. The rmsd was 1.92 ( 0.61 Å, thus indicating quite a wide fluctuation over time. The radius of gyration of the average D system is comparable to those obtained for systems A-C, but inspection of the variation of the radius versus time clearly indicated a steady increase starting from t ) 1.3 ns until the end of the simulation (Figure 2). This increase is associated to an expansion of the apo system D. More insights into the selective conformational changes which occurred in system D come from inspection of Figure 3 which shows the average fluctuation per residue, calculated as time-average rmsd from each of the four average MD structures. Assignment of each residue to secondary structure elements indicated that most of the conformational changes taking place in system D resided in the C-terminus of the protein, and in particular in the region of the loop between helices 11 and 12 (H11-H12 loop) and of helix 12 (H12). Thus, examination of the time averaged structures of complexes A-D revealed a significant similarity to the crystal structure when the ligand or the coactivator

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Figure 3. Residue fluctuation from the average MD structures for the four simulations A-D. Graphical representation of secondary structure elements is given in correspondence to residue number. The highest rmsd (Å) is found in system D for residues 445464, belonging to loop H11-H12 and helix H12. ‘C2’ and ‘D2’ indicate coactivator 1 and coactivator 2.

Figure 4. (Left) Variation of the rmsd (Å) of helix 12 (H12) during the 3 ns trajectory. While H12 was absolutely stable during the simulation in system A-C, a major deviation took place for system D. (Right) Graphical representation of the AF2 (H12) in the crystal structure (green) and at the end of the dynamics simulation for system D (red).

peptides are present (systems A-C). On the contrary, when the apo-LBD of FXR (system D) is simulated starting from the crystal structure of the holo-LBD, a major conformational change did occur. A more detailed analysis of the four simulations is reported below. 2. Activation Function-2 (AF2, Helix 12). In systems A, B, and C the crucial helix H12 remained in the active conformation along all the 3 ns simulations. The rmsd of H12 computed over the backbone atoms of residues Thr459-Asp467 was 1.34 ( 0.14 Å, 1.20 ( 0.21 Å, and 1.55 ( 0.32 Å for systems A, B, and C, respectively. In system D, on the contrary, a major conformational change was observed for H12. The rmsd for H12 with respect to the crystal structure was, in fact, 3.96 ( 2.14 Å. Figure 4 clearly indicates the movement of H12 from the crystal structure position. These results point out that the LBD of FXR is unable to maintain H12 in the active, ‘holo-like’ conformation in the absence of the ligand and of the coactivator peptides. 3. Features Affecting H12 Conformational Stability. In system A, stabilization of H12 is due to the simultaneous role played by (i) the activation trigger between tryptophan 466 (W466) and histidine 444 (H444); and (ii) by the highly conserved interaction

(referred to as ‘charge-clamp’, see below) between lysine 318 (K318), glutamate 464 (E464), and the coactivator peptide (Figure 5). The first effect is directly mediated by the ligand. The presence of ring A of 6ECDCA constrains H444 to its ‘active’ disposition, with the N atom pointing toward the electron rich aromatic system of W466. In our simulations A and B, the ligand participated in H12 stabilization by forming two hydrogen bonds between the Y358 OH group (donor) and the 6ECDCA 3-OH group (acceptor) and between the 3OH group of 6ECDCA (donor) and the Nδ of H444. It has also been argued that in the absence of the 3OH group (like in the case of 3DCDCA) Y358 could act as a donor in the formation of the hydrogen bond with H444. The hydrogen bond pattern above-described and observed in the crystal structure is maintained along the whole simulation when system A is studied. In particular, Figure 6 shows that the hydrogen bond between the 3-OH (donor) group of 6ECDCA and the H444 Nδ atom (acceptor) is stable during almost all the simulation, with an average distance of 2.21 ( 0.31 Å. Similarly, the Y358 OH group (donor) forms another hydrogen bond with the 3-OH group (acceptor) of 6ECDCA and this interaction remains stable (d ) 1.93 ( 0.17 Å) during all the simulation. The hydrogen bond



Figure 5. (Left) Detailed view of the activation trigger and the charged clamp in the stabilization of AF2 (red) in the crystal structure of ECDc. H444 N atom points toward the center of W466, while the charged clamp (K318 and E464) interacts with the coactivator (green). (Right) Detailed view of the hydrogen bonds formed between the 6ECDCA 3OH group and H444 and Y358. These interactions restrict the mobility of H444 and stabilized the trigger.

Figure 6. Variation of selected distances defining the hydrogen bond pattern involved in the interaction between the 6ECDCA 3OH group, His444, and Tyr358. (a) The hydrogen bond between the 3-hydroxy group of 6ECDCA and H444 is stable during the whole simulation in system A, while it stands until t ) 2.6 ns for system B. (b) In system A, the hydrogen bonding between the 3-hydroxy group of 6ECDCA (donor) and the hydroxy group of Y358 (acceptor) is never formed. In system B, at t ) 2.6 ns, there is formation of this interaction. (c) In system A, the hydrogen bonding between the 3-hydroxy group of 6ECDCA (acceptor) and the hydroxy group of Y358 (donor) is present during the whole simulation. In system B, at t ) 2.6 ns, there is a breaking of such interaction as a consequence of the above redistribution of hydrogen bonding character of the pair Y358-3OH of 6ECDCA. (d) Neither in system A nor in system B there is formation of hydrogen bonding between Y358 and H444. (e) When the ligand is removed (systems C and D) the only potential hydrogen bonding partners are Y358 and H444, but the formation of hydrogen bonding is loose over the simulation time with very high fluctuations.

between 3OH group (donor) and the Y358-OH group (acceptor) is never formed. The hydrogen bond between Y358 and H444 is likewise never formed, the average

distance being 3.56 ( 0.33 Å. Also in simulation B, when the ligand but not the coactivators are present, there is a strong stabilization of the trigger (Figure 6) and the

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Figure 7. (Left) Variation of the distance (Å) between the CR atoms of K318 and E464. The distance is very stable during the 3 ns simulation time for systems A-C, comparable with that of the crystal structure. The distance is also stable for system D until t ) 1.6 ns, when H12 starts its displacement (see also Figure 1). From t ) 1.6 to t ) 3.0 ns, a major increase of the distance is seen for system D. (Right) Variation of the distance (Å) between the N atom of K318 and the Cδ atom of E464. The distance is stable during the 3 ns simulation time for systems A-C and indicates that no salt bridge is formed. In the case of system D, the salt bridge is formed during the heating phase and it stands until disruption of the helix 12 at t ) 1.6 ns.

hydrogen bonding pattern between ligand, Y358, and H444 is maintained until t ) 2.6 ns, when the 3OH group (donor) rotates and forms a hydrogen bond with the Y358-OH group (acceptor), the distance during the last 0.4 ns being 1.91 ( 0.15 Å. Again, the hydrogen bond between Y358 and H444 is never formed. The average distance between the hydrogen atom of the hydroxy group of Y358 and the Nδ atom of H444 is of 4.23 ( 1.09 Å. Profoundly different is the situation when systems C and D (no ligand present) are analyzed. In both cases, the only potential hydrogen bonding partners are Y358 and H444. Indeed, the Y358-OH group formed the hydrogen bond with the H444 Nδ atom, but this interaction was very loose, as the distance between both atoms fluctuated widely (Figure 6). The average value for the distance is 3.14 ( 1.50 Å for system D and 3.35 ( 1.46 Å for system C. Nevertheless, despite the lack of trigger stabilization, helix H12 was stable in simulation C. H12 stabilization is achieved by physical constraint imposed by the presence of the coactivator peptide through the second general mechanism of stabilization, i.e., the formation of the charge clamp between E464 (H12), K318(H3), and coactivator peptide (see below). Finally, in the case of simulation D (no ligands nor coactivator) the loss of the trigger interaction is accompanied by disruption of the active conformation of H12. 4. Features Affecting the Recognition of the Canonical Coactivator Peptide. The recognition between the LxxLL motif of coactivator peptides and NRs is mediated by hydrophobic interactions and by a charge clamp constituted by a lysine residue positioned in helix 3 and a glutamic acid residue localized in helix 12.3 The γ-carboxylate of E464 (H12) binds the backbone amide of the N-terminal domain of the coactivator while a hydrogen bond is formed between the -amino group of K318 (H3) and the carbonyl oxygen atom of the carboxy terminus of the peptide. Figure 7a shows the distances in function of the simulation time for the four simulations. In the case of systems A-C, the distance between the CR atoms of E464 and K318 remained very stable during the simulation (d ) 7.42 ( 0.34 Å, d ) 8.42 ( 0.34 Å, d ) 7.90 ( 0.37 Å, respectively) In the case of system D, the distance started increasing at t ) 1.6 ns and grew up until the end of the

simulation (d ) 10.60 ( 2.59 Å). This occurred as a result of the displacement of H12 above-described. Of interest is the examination of Figure 7b, where the distance between the side chains of E464 and K318 is reported as a function of the simulation time. Again, a very stable trajectory was observed for systems A-C (d ) 7.50 ( 0.45 Å, 7.15 ( 0.90 Å, and 6.98 ( 0.41 Å, respectively), while a major displacement occurred in system D. In this case, however, the stability of systems A-C deserves some comments. While in systems A and C the invariance of the distance between Cδ of E464 and N of K318 is clearly due to the presence of the coact1 peptide tightly bound to these residues, in system B one could have expected the formation of a salt bridge between lysine and glutamate. In our condition, this did not occur for at least 3 ns. In contrast, when system D is analyzed, the salt bridge is immediately formed during the heating phase and is kept until H12 relocated (t ) 1.6 ns). The apparent conclusion that can be drawn from these results is that the ligand (simulation B) prevents the formation of the salt bridge, thus preparing the canonical crevice for the binding of the coactivator. Being aware of the potential relevance of this finding, we tested the possibility that the formation of salt bridge in simulation D was obtained by chance during the heating phase. Thus, several additional “D” simulations were started, and in four out of the five new simulations there was formation of the salt bridge in the heating phase, while in the fifth the formation took place at t ) 0.2 ns in the equilibration phase. Notably, when additional simulations were carried out for system B, formation of the salt bridge occurred only once in five independent simulations. We are therefore quite confident in the reliability of the results, and it turned out that the intimate role of ligand binding to LBD is that of making room for the coactivator. It is also worth noting that a survey of the Brookhaven Protein Database (PDB) identified 35 NR structures complexed with an agonist molecule and no coactivator. Of these, 13 (representative of six different receptors) have the crevice open, while 22 (but representative of only three receptors) have the salt bridge formed. 5. Features Affecting the Stability of the Canonical Coactivator Peptide. Crystallization of the LBD or FXR complexed with 6ECDCA produced a dimeric structure in which one of the two monomers had two peptides bound to the receptor while the other one



Figure 8. (Left) rmsd (Å) variation for the canonical LxxLL peptide during the 3 ns simulation time. The peptide is very stable in the case of system A (ligand present). The peptide remains bound also to system C (no ligand), but a larger fluctuation is observable. (Right) rmsd (Å) variation for the noncanonical second LxxLL peptide during the 3 ns simulation time. The peptide is rather stable in system C, whereas it is released from the LBD in system A. Note the difference in the y-scale between the two plots.

Figure 9. (Left) Unfolding of the noncanonical peptide and relocation of the H1-H2 loop in simulation A: Dark, t ) 3.0 ns; gray, crystal structure. Note the invariability of the 6ECDCA ligand. (Right) The same as above, side view.

showed only one peptide in the canonical crevice.17 Hypotheses have been made on the role of this second peptide.21 One of the aims of our present study was to clarify this important issue. Figure 8a shows the rmsd for the canonical coactivator (coact1) in system A and system C. In both cases the rmsd was low and visual inspection of the average MD structures revealed that the peptide remained bound to the receptor for the whole simulation. A potentially relevant observation is, however, that coact1 has a significantly lower fluctuation in the case of system A with respect to system C (rmsd ) 1.26 ( 0.19 Å, 1.56 ( 0.49 Å, respectively). This adds further support to the notion that the ligand is necessary to maintain the peptide tightly and constantly bound to the LBD. 6. The Second LxxLL Peptide Is Stably Bound in the Absence of the Ligand But Is Released in Its Presence. Much more surprisingly for us was the inspection of Figure 8b, where the rmsd of the second peptide (coact2) is shown as a function of the simulation time. For both systems A and C, a looser binding was observed (rmsd ) 3.47( 0.76 Å; 1.82 ( 0.27 Å, respectively) with respect to coact1. Visual inspection of the average MD structures indicated that coact2 was stably bound to system C while clearly destabilized by the presence of the ligand in system A. 7. A Microdevice Formed by Three Amino Acids Is Responsible for the Dissociation of the Second Peptide. In particular, when simulation A is examined, the microdevice responsible for coact2 dissociation could

Table 3. Dynamic Changes of the M262-P263-Q264 Microdevice Compared with the Crystal Structures structure chain A (1OSV) chain B (1OSV) A (average structure) C (average structure)

Met262 Pro263 Gln264 Met262 Pro263 Gln264 Met262 Pro263 Gln264 Met262 Pro263 Gln264

PHI

PSI

-61.1 -55.6 -59.9 35.4 -50.7 81.0 -57.9 ( 15.7 -49.2 ( 9.4 -61.2 ( 21.0 -159.5 ( 29.8 -66.5 ( 13.1 -74.8 ( 27.9

135.8 139.5 -34.3 41.9 178.5 92.2 148.3 ( 15.7 114.6 ( 7.7 -22.8 ( 25.4 82.9 ( 16.9 85.3 ( 8.8 145.0 ( 18.0

be identified in the stretch M262-P263-Q264 present in the loop H1-H2. M262 is in hydrophobic contact with the carboxylic tail of 6ECDCA, while Q264 is hydrogen bonded to Asn-2′ of coact2. The presence of the ligand in simulation A had the effect of modifying the psi-phi angle of M262 with respect to the crystal structure (psix-ray ) 41.8°, psiaverageMD ) -57.9 ( 15.7°; phix-ray ) 41.9°, phiaverageMD ) 148.9 ( 15.7°). This movement was transmitted to Q264 through the rigid P263 and resulted in the loss of the hydrogen bond between Q264 and coact2 (Figure 9). Interestingly, the average MD structure of system A, where the second peptide has been released, is very similar to the crystal structure of chain A of 1OSV, where only one peptide could be crystallized (Table 3). Thus, the stabilization of coact2 is mediated by the M262-P263-Q264 microdevice. There are two competing interactions acting on the microde-

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vice. The first one is the stabilization of M262 by the agonist 6ECDCA; the other one is the stabilization of Q264 by Ans-2′ of coact2. In simulation A, the interaction with 6ECDCA is predominant and causes the redistribution of P263 and Q264 torsional angles, thus inducing the release of coact2. In simulation C, Q264 interacts with coact2, stabilizes it, and forces the backrelocation of psi and phi angles of P263 and M262. 8. Role of the Second LxxLL Peptide: Artifact or Potential Biological Significance? According to our simulation, the structure frozen in chain B of 1OSV seems to be a metastable state of the complex between LBD, ligand, and peptide. The stable state is represented by chain A, to which chain B evolves and in which only the canonical peptide is bound. Thus, the quaternary complex represented by chain B of 1OSV is likely to be a crystallographic artifact as such, but the results from our simulation C do not rule out the possibility that an enhanced coactivator recruitment could be achieved through the second, noncanonical crevice localized close to loop H1-H2.

2. The Interaction between H444 and W466 Is Not Sufficient To Stabilize H12 in the Active Conformation. In the absence of of both the ligand and the coactivator, the potential hydrogen bond interaction between Y358 and H444 is not sufficient to stabilize the trigger, and the interaction between H444 and W466 became rapidly ineffective. So, what is the role of the H444-W466 interaction? A possible scenario is that the loop H11-H12 controls the flexibility of H12, and the loop is intrinsically unstable in the active conformation. The stabilization of the active conformation of the LBD requires stabilization of the loop rather than of H12, and this is achieved primarily through physical constraint of H444. This explanation accounts for the agonist activity of compounds such as GW4064, fexaramine, and 3-deoxy-CDCA which are intrinsically unable to participate in the hydrogen bonding pattern described above. The ‘fine-tuning’ rather than ‘true activation’ role played by the H444-W466 interaction also accounts for the apparent lack of constitutive activity of FXR. Indeed, even if one assumes that there exists a conformational equilibrium between the ‘active’ and the ‘inactive’ conformation of H12, the active conformation cannot be stabilized in the absence of the ligand. It is possible to argue, from our simulation C, that the ‘active’ conformation can in principle be recognized by the coactivator peptide, and this is sufficient, according to our results, to stabilize H12. However, in the absence of the bile acid ligand, the canonical crevice for the coactivator is inaccessible because of the formation of the salt bridge between K318 and E464. 3. MD Simulations Point Out an Unprecedented Role for Loop H1-H2 and Support a Potential Biological Role for the Second Coactivator Binding Crevice. The most intriguing feature that emerged from the solution of the X-ray structure of the holo-LBD of FXR was the presence of two coactivator peptides. One open question is the physiological/structural role, if any, of the second peptide. Our results converge into the notion that the presence of the second peptide may have biological significance although the quaternary complex ‘frozen’ in chain B of 1OSV is most likely to represent a metastable state due to a crystallographic artifact. In particular, it has been proposed that the second binding site represents an “on deck” site for the LxxLL, which can flank the canonical docking site into the heterodimer partner.21 Our MD simulations give support to this hypothesis, and the following observations are of particular interest. (i) The ligand, and the carboxylic tail of 6ECDCA in particular, operates a microdevice (composed of the M262-P263-Q264 stretch) which is responsible for the primary dissociation of coact2 from the LBD. (ii) The canonical peptide plays also a role in this game since the binding of the first LxxLL fragment directly affects helix H3 which is in contact with P263 through I294. Thus, both the ligand and the first coactivator cooperate in ‘removing’ loop H1-H2 accessibility to the second peptide. A dynamic situation can be envisaged where a LxxLL box can be bound to the second docking site in a ‘resting’ position. Binding of the ligand and recruitment of the ‘canonical’ peptide induce the molecular signals leading to the dissociation of the second box and to its possible interaction with the heterodimer partner.21 The peculiar

Discussion Medium-range molecular dynamics simulations have been productively applied to the study of the interaction between several nuclear receptors and their ligands including the vitamin D receptor,22 glucocorticoid receptor,23 estrogen receptor,24 progesterone receptor,25 and androgen receptor.26 In particular, examination of the dynamic behavior of these complexes has often resulted in an experimentally checkable hypothesis on the molecular basis responsible for their pharmacology. The case of the FXR receptor is particularly suited to MD analysis in light of the unusual features that have emerged from the resolution of the X-ray structure of the holo-LBDs. In this paper we report, for the first time to our knowledge, a MD study aimed not only at understanding the mutual interaction between the ligand and the LBD, but also at disclosing the dynamic pattern of interactions leading to coactivator stabilization. 1. The Binding of the Agonist Stabilizes H12 in the Active Conformation and Makes Room for Coactivator Binding. The bile acid agonist 6ECDCA stabilizes the holo conformation of the FXR LBD. Indeed, when the binary complex between the LBD of FXR and 6ECDCA was simulated, the H12 helix was kept into the active conformation through the stable formation of a pattern of hydrogen bonds involving the 3-hydroxy group of 6ECDCA, the Y358-OH and H444. In particular, the interaction between the 3-hydroxy group of 6ECDCA and H444 was stable as long as 2.6 ns. Interestingly, in the binary complex B the stabilization of H12 was not accompanied by the formation of a salt bridge between K318 and E464, and the canonical crevice for coactivator binding remained accessible for the whole 3 ns simulation time. Thus, the presence of the agonist seemed to be mandatory for subsequent coactivator binding. The formation of the above-described hydrogen bonding pattern is however neither required nor sufficient to induce H12 stabilization since, if H12 is physically constrained to its active conformation, as in system C, there is stabilization of the H444W466 interaction even if the hydrogen bonding pattern between Y358 and H444 is very loose.



role as a molecular switch for loop H1-H2 is confirmed by other observations. First, the crystal structure of FXR LBD complexed with two peptides17 has unusual values of the psi-phi angles for the M262-Q263 stretch. The same fragment is disordered in the crystal structure of hFXR LBD bound to fexaramine (with no peptide bound).9 Similarly, the loop is disordered in the RXR holo structure but not in the apo form.27 Thus, the loop H1-H2 seems to be endowed with a peculiar flexibility and to be suited to respond to binding of ligands and peptides in such a way to operate signaling for the subsequent steps of cofactor recruitment and gene expression. In conclusion, nanosecond time scale MD simulation showed a complex and dynamic pattern of interaction between the LBD of FXR, a bile acid agonist, and LxxLL peptides which were not immediately apparent in the crystal structure of these systems. Response to ligand binding is mediated and transmits far from the binding site, through microdevices mainly positioned in the loops H1-H2 and H11-H12. These findings may have a relevance for the further understanding of the molecular functioning of NRs and for the design of novel FXR modulators.

(9) Downes, M.; Verdecia, M. A.; Roecker, A. J.; Hughes, R.; Hogenesch, J. B.; Kast-Woelbern, H. R.; Bowman, M. E.; Ferrer, J. L.; Anisfeld, A. M.; Edwards, P. A.; Rosenfeld, J. M.; Alvarez, J. G.; Noel, J. P.; Nicolaou, K. C.; Evans, R. M. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol. Cell. 2003, 11, 1079-92. (10) Chen, W.; Owsley, E.; Yang, Y.; Stroup, D.; Chiang, J. Y. Nuclear receptor-mediated repression of human cholesterol 7alphahydroxylase gene transcription by bile acids. J. Lipid Res. 2001, 42, 402-12. (11) Goodwin, B.; Jones, S. A.; Price, R. R.; Watson, M. A.; McKee, D. D.; Moore, L. B.; Galardi, C.; Wilson, J. G.; Lewis, M. C.; Roth, M. E.; Maloney, P. R.; Willson, T. M.; Kliewer, S. A. A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell. 2000, 6, 517-526. (12) Lu, T. T.; Makishima, M.; Repa, J. J.; Schoonjans, K.; Kerr, T. A.; Auwerx, J.; Mangelsdorf, D. J. Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell 2000, 6, 507-515. (13) Willson, T. M.; Jones, S. A.; Moore, J. T.; Kliewer, S. A. Chemical genomics: functional analysis of orphan nuclear receptors in the regulation of bile acid metabolism. Med. Res. Rev. 2001, 21, 513522. (14) Francis, G. A.; Fayard, E.; Picard, F.; Auwerx, J. Nuclear receptors and the control of metabolism. Annu. Rev. Physiol. 2003, 65, 261-311. (15) Chen, W.; Owsley, E.; Yang, Y.; Stroup, D.; Chiang, J. Y. Nuclear receptor-mediated repression of human cholesterol 7alphahydroxylase gene transcription by bile acids. J Lipid Res. 2001, 42, 1402-12. (16) Lew, J. L.; Zhao, A.; Yu, J.; Huang, L.; De Pedro, N.; Pelaez, F.; Wright, S. D.; Cui, J. The farnesoid X receptor controls gene expression in a ligand- and promoter-selective fashion. J Biol. Chem. 2004, 279, 8856-61. (17) Mi, L. Z.; Devarakonda, S.; Harp, J. M.; Han, Q.; Pellicciari, R.; Willson, T. M.; Khorasanizadeh, S.; Rastinejad, F. Structural basis for bile acid binding and activation of the nuclear receptor FXR. Mol. Cell. 2003, 11, 1093-100. (18) Costantino, G.; Macchiarulo, A.; Entrena-Guadix, A.; Camaioni, E.; Pellicciari, R. Binding mode of 6ECDCA, a potent bile acid agonist of the farnesoid X receptor (FXR). Bioorg. Med. Chem. Lett. 2003, 13, 1865-8. (19) Kale´, L.; Skeel, R.; Bhandarkar, M.; Brunner, R.; Gursoy, A.; Krawetz, N.; Phillips, J.; Shinozaki, A.; Varadarajan, K.; Schulten, K. NAMD2: Greater Scalability for Parallel Molecular Dynamics. J. Comput. Phys. 1999, 151, 283-312. (20) Cerius-2 and Insight-II, Accelrys, San Diego, CA. (21) Nettles, K. W.; Greene, G. L. Nuclear receptor ligands and cofactor recruitment: is there a coactivator “on deck”? Mol. Cell 2003, 11, 850-1. (22) Gonzalez, M. M.; Samenfeld, P.; Perakyla, M.; Carlberg, C. Corepressor excess shifts the two-side chain vitamin D analog Gemini from an agonist to an inverse agonist of the vitamin D receptor. Mol. Endocrinol. 2003, 17, 2028-38. (23) Stockner, T.; Sterk, H.; Kaptein, R.; Bonvin, A. M. Molecular dynamics studies of a molecular switch in the glucocorticoid receptor. J. Mol. Biol. 2003, 328, 325-34. (24) Nam, K.; Marshall, P.; Wolf, R. M.; Cornell, W. Simulation of the different biological activities of diethylstilbestrol (DES) on estrogen receptor alpha and estrogen-related receptor gamma. Biopolymers 2003, 68, 130-8 (25) Hillisch, A.; von Langen, J.; Menzenbach, B.; Droescher, P.; Kaufmann, G.; Schneider, B.; Elger, W. The significance of the 20-carbonyl group of progesterone in steroid receptor binding: a molecular dynamics and structure-based ligand design study. Steroids 2003, 68, 869-78. (26) Wu, J. H.; Gottlieb, B.; Batist, G.; Sulea, T.; Purisima, E. O.; Beitel, L. K.; Trifiro, M. Bridging structural biology and genetics by computational methods: an investigation into how the R774C mutation in the AR gene can result in complete androgen insensitivity syndrome. Hum. Mutat. 2003, 22, 465-75. (27) Egea, P. F.; Mitschler, A.; Rochel, N.; Ruff, M.; Chambon, P.; Moras, D. Crystal structure of the human RXRalpha ligandbinding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J. 2000, 19, 2592-601.

Acknowledgment. A.E.G. gratefully acknowledges the “Secretaria de Estado de Universidades y Educacion” of Spain for financial support. The authors gratefully acknowledge the “Centro de Servicios de Informa´tica” of the University of Granada (Spain) for the generous allowances of computer time on their SGI Origin 3400 computer. References (1) Willson, T. M.; Moore, J. T. Genomics versus orphan nuclear receptorssa half-time report. Mol. Endocrinol. 2002, 16, 113544. (2) Wurtz, J. M.; Bourguet, W.; Renaud, J. P.; Vivat, V.; Chambon, P.; Moras, D.; Gronemeyer, H. A canonical structure for the ligand-binding domain of nuclear receptors. Nat. Struct. Biol. 1996, 3, 87-94. (3) Savkur, R. S.; Burris, T. P. The coactivator LXXLL nuclear receptor recognition motif. J. Pept. Res. 2004, 3, 207-12. (4) Wang, H.; Chen, J.; Hollister, K.; Sowers, L. C.; Forman, B. M. Endogenous bile acids are ligands for the nuclear receptor FXR/ BAR. Mol. Cell. 1999, 3, 543-53. (5) Parks , D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.; Consler, T. G.; Kliewer, S. A.; Stimmel, J. B.; Willson, T. M.; Zavacki, A. M.; Moore, D. D.; Lehmann. J. M. Bile acids: natural ligands for an orphan nuclear receptor. Science 1999, 284, 13658. (6) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R. M.; Luk, A.; Hull, M. V.; Lustig, K. D.; Mangelsdorf. D. J.; Shan, B. Identification of a nuclear receptor for bile acids. Science 1999, 284, 1362-5. (7) Pellicciari, R.; Fiorucci, S.; Camaioni, E.; Clerici, C.; Costantino, G.; Maloney, P. R.; Morelli, A.; Parks, D. J.; Willson, T. M. 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity. J. Med. Chem. 2002, 45, 3569-72. (8) Maloney, P. R.; Parks, D. J.; Haffner, C. D.; Fivush, A. M.; Chandra, G.; Plunket, K. D.; Creech K. L.; Moore, L. B.; Wilson, J. G.; Lewis, M. C.; Jones, S. A.; Willson, T. M. Identification of a chemical tool for the orphan nuclear receptor FXR. J. Med. Chem. 2000, 43, 2971-4.

JM049182O

Molecular Dynamics Simulation of the Ligand Binding Domain of

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