proteins STRUCTURE O FUNCTION O BIOINFORMATICS

Computational studies of LXR molecular interactions reveal an allosteric communication pathway Sofia Burendahl and Lennart Nilsson*

Department of Biosciences and Nutrition, and Center for Biosciences, Karolinska Institutet, SE-141 83 Huddinge, Sweden

ABSTRACT INTRODUCTION

The liver X , LXRa, is an important Nuclear receptors (NRs) are transcription factors that regulate regulator of involved in metabolism expression in response to a signal. The nature of the given signal can and inflammation. The mechanism of com- vary although many NRs respond to a small molecule like a hormone or munication between the cofactor peptide a metabolic intermediate. The ligand binding can be specific and with and the ligand in the ligand-binding pocket high affinity, but the NRs can also work as molecular sensors with lower is a crucial and often discussed issue for the affinity for the ligand and with less selectivity. NRs that function as met- nuclear receptors (NRs), but such allosteric abolic sensors often form heterodimers with (RXR), signaling pathways are difficult to detect organizing the response of nutrition.1,2 and the transmission mechanism remains The liver X receptors, LXRa (NR1H3) and LXRb (NR1H2), are NRs elusive. Here, we apply the anisotropic ther- that respond to oxidized forms of cholesterol (oxysterols) and regulate mal diffusion method to the LXRa with genes involved in lipid and cholesterol homeostasis (metabolism).3–5 bound coactivator and ligand. We detected a Identification of selective LXR nonsteroid ligands T-09013176 and possible communication pathway between 5 the coactivator peptide and the ligand. The GW3965 provides an opportunity to explore LXR biology and possibil- 7 signal is transmitted both through the re- ities for a therapeutical development (reviewed in Ref. ). Recent studies ceptor backbone and side chains. A key sig- point out LXR as a target for treatment of atherosclerosis, type 2 diabe- naling residue is the first leucine in the tes, inflammation, and neurodegenerative diseases.8 cofactor peptide recognition motif LXXLL, The two LXR subtypes are encoded by different genes and share about which is conserved within the NR cofactors, 75% amino acid identity in the ligand-binding domain (LBD). LXRb is suggesting a general mechanism for alloste- expressed in all tissues, but LXRa expression is restricted to the liver, ric signaling. Furthermore, we studied the small intestine, spleen, kidney, adrenal gland, adipose tissue, and macro- LXR receptor and cofactor molecular inter- phages. LXRa knockout in mice results in accumulation of cholesterol actions in detail using molecular dynamics esters in liver, due to impaired synthesis. Liver expressed LXRb simulations. The protein–protein interaction does not compensate for the loss of LXRa activity, which suggests differ- patterns in the complexes of nine different 9 a cofactor peptides and holo-LXRa were char- ent biological mechanisms for the two LXR subtypes. Although LXR b acterized, revealing the importance of the activation causes an unwanted increase of triglyceride levels, LXR has receptor–cofactor charge clamp interaction. an important role in lipid efflux through increased expression of the Specific, but infrequently occurring interac- LXR target genes of the transport protein ABCA1.10 Thus, specificity tions were observed toward the cofactor between the receptors subtypes has to be considered for a beneficial LXR peptide C-terminal residues. Thus, addi- effect. Due to the high structural resemblance specificity has turned out tional specificity between LXRa and its to be a challenging problem for LXR drug development. The interior of cofactors is likely to be found in molecular the ligand-binding pocket (LBP) is comprised of 31 identical amino acids interactions outside the cofactor peptide or and a distance of 6 A˚ from any ligand is needed to diverge the subtypes in other biological factors.

Proteins 2012; 80:294–306. Burendahl’s current address is Computational Biology Research Center, National Institute of Advanced VC 2011 Wiley Periodicals, Inc. Industrial Science and Technology (AIST), AIST Tokyo Waterfront BIO-IT Research Building, 2-4-7 Aomi, Koto-ku, 135-0064 Tokyo, Japan Additional Supporting Information may be found in the online version of this article. Key words: ; liver X recep- Grant sponsor: Swedish Research Council tor; molecular dynamics; allosteric signaling; *Correspondence to: Lennart Nilsson, Department of Biosciences and Nutrition, Karolinska Institutet, SE- ligand binding. 141 83 Huddinge, Sweden. E-mail: [email protected] Received 1 August 2011; Revised 9 September 2011; Accepted 22 September 2011 Published online 29 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/prot.23209

294 PROTEINS VC 2011 WILEY PERIODICALS, INC. LXR Allosteric Communication Pathway

Figure 1 Schematic picture of LXR LBD. (a) The NR domain organization (A–F) includes the DNA-binding domain (DBD) and the ligand-binding domain (LBD). Four structurally distinct but functionally couple regions have an allosteric communication. (b) The LBD’s three layered (black, gray, and white) a-helical sandwich includes two b-sheets (S1 and S2) and 12 helices (h1–h12), where h2 is unfolded and forms the h2 loop. The enclosed LBP stretches from h12 to the b-sheets. A ligand bound can interact with residues from h1, h2 loop, h3, h5, h7, h11, h12, and the b-sheets. This picture was made based on the structure of the minimized 1UHL model. Ligand area was defined as the vdW surface of the bound T-17 ligand. from each other.11 The LBP of LXR is mainly of a On activation LXR forms a heterodimer together with hydrophobic character, but it does contain a few polar or RXR.13 The LXR–RXR dimer can be activated exclusively charged residues. The LBP stretches from the helix 12 by an RXR agonist, an LXR agonist or by both. The per- (h12; NRs have twelve helices, h1–12) to the b-sheets missive action of the LXRs suggests communication (Fig. 1). Structural features such as the position of the h2 pathways in the heterodimer complex often described as loop and the possibility to extend the pocket toward the allosteric effects. Allosteric interactions involve communi- adjacent solvent cavity provide LXR with the possibility cation between (distal) sites on proteins and have been to bind chemically and structurally diverse ligands, with- found to be fundamentally important for many biological out affecting the overall receptor structure. Although the processes the classical example being the oxygen binding h2/b-sheet side of the LBP displays a high degree of flex- of hemoglobin.14 In the NRs the allosteric network con- ibility the side toward h12 is more rigid. The exterior of nects four structurally distinct but functionally coupled h12 forms, together with residues from h3 and h4, the regions of the RXR heterodimer complex: the ligand- activation function 2 (AF-2) surface where cofactors binding site, the cofactor binding surface, the AF-2 site, bind. Several cofactors, often binding the receptor with and the dimerization surface between the receptors (Fig. low affinity, have been identified for LXR. The strength 1). To understand the allosteric mechanism one has to of the binding differs between different cofactors and can identify the residues which participate in the communi- also be modified with different ligands.12 Thus, a low-af- cation path. Methods like the statistical-coupling analysis finity interaction can be altered, and new interactions (SCA)15,16 identify networks of (energetically) coupled between the receptor and the cofactors can form. Rela- residues, by statistical analysis of coevolutionary con- tively little is known about the interaction between LXR straints on a protein family. This sequence based analysis and its cofactors on a molecular level. To gain insight has been shown to agree with mutagenesis studies and into the interactions between the LXR receptor and its known allosteric mechanisms, and can be used as an in- known cofactors, we performed molecular dynamics sim- dication of which amino acids are participating in the al- ulations of the LXRa–RXRb complex, with nine different losteric signaling. Molecular dynamics simulation and cofactor peptides including both coactivator and core- analysis of principal components and correlated move- pressor peptides, bound to LXRa. The aim is to charac- ments has also been used to increase the understanding terize the interaction and identify specific interactions of allosteric mechanisms. In a study of c-Src a regulative important for the binding affinity. Tyr phosphorylation 40 A˚ from the catalytic site caused a

PROTEINS 295 S. Burendahl and L. Nilsson change in the dynamics of the SH2 and SH3 domains as speed up the calculation of nonbonded interactions revealed by molecular dynamics studies.17 Other compu- where appropriate.29 The modeled complex of LXR–RXR tational approaches like normal mode analysis have also was based on the coordinates of the PDB id 1UHL,30 turned out to be useful in allosteric investigations.18 Fur- which includes human LXR-a residues 206–447, human thermore, experimental work using for example NMR or RXR-b residues 298–553, two 10 amino acid peptides of X-ray techniques indicate that changes of dynamic prop- NcoA2-2, the RXR ligand Methoprene acid and the syn- erties are involved in allosteric signaling.19–23 Nonethe- thetic LXR agonist T-0901317 (T-17). The different coac- less, a continuous allosteric communication pathway tivator peptide sequences of NCOA2-1,2,3 (also called between distant sites in the NRs has not been identified SRC2, TIF2, and GRIP1), NCOA6-1,2 (RAP250 and and the mechanism of transmission of the physical signal ASC2) and corepressor peptide sequences PROX1-1,2 remains elusive. and NR0B2-1,2 (SHP) were modeled into the complex Recently studies of PDZ-domain proteins showed that by using the backbone coordinates of the peptide of the the transmission of an intramolecular signal can be NCOA2-2 X-ray structure, and the side chains were detected on a picosecond timescale using anisotropic ther- added in an extended conformation. The LXR and RXR mal diffusion (ATD).24 In the ATD method, the system is backbone of residues with lacking electron densities was initially cooled, so that the normal thermal fluctuations in modeled with the Swiss PDB modeler (swissmodel. the protein become minimal, and then a selected region of expasy.org). After a structural visualization all histidines the system is coupled to a heat bath. The accumulated were assigned a neutral state, protonated at the Ne2 heat will gradually propagate throughout the system away atom. Hydrogen atoms were added as previously from the higher temperature region. Heating at a ran- described for CHARMM.31 The ligand parameters were domly selected site causes a redistribution of the heat to selected from similar groups in the CHARMM all-atom the closest environment without any specific direction, but protein and lipid parameter files.27,32–34 heating at certain amino acids can cause a transmission The complex was solvated with a 10 A˚ thick shell of through a set of amino acids to a distal site. Such transmis- TIP3P35 water molecules, including crystallographic sion takes place along an energetically favorable pathway, waters, before the structure was relaxed to the which in PDZ was found to correspond to an allosteric sig- CHARMM22 all-atom force field34 by 500 steps of the naling pathway.24 After the transmission of the thermal adopted-basis Newton–Raphson minimization method signal, one may be able to define an intramolecular signal- with restraints on the a-carbons and 1500 steps without ing pathway and discuss the physical transmission mecha- any restraints. The solvent molecules were kept from nism. Here, we apply the ATD method on LXR to study escaping by a mass-weighted quartic potential with force 2 2 2 the signaling between the cofactor peptide and the ligand constant 4.165 3 10 8 kcal mol 1 A˚ 4, presenting an in the LBP. The aim is to identify amino acids participating initially flat restraint energy that for water oxygens reach 2 in an allosteric pathway between these regions. 1 kcal mol 1 at 35 A˚ from the system center of mass The Other modes of communication within the protein, nonbonded energies and forces were smoothly shifted to possibly involving larger and slower conformational rear- zero at 12 A˚ inter-atomic separations. The nonbonded rangements of, for instance, helices in the LBP, would list was updated heuristically using a 14 A˚ cutoff. The not be detected by the approach we have chosen in this temperature was set to 300 K Æ10 K. SHAKE36 was used study. Instead we focus on changes in dynamic properties to constrain X–H bonds during the MD simulations. like atom fluctuations and conformational changes in Data was collected for 10 ns with a time step of 2 fs. side chains. This is in line with a new view of protein ATD24 simulations were performed on LXR. For this allostery, where allostery is regarded as a thermodynamic approach the LXR with ligand T-17 and NCoA2-2 cofac- phenomena including both enthalpic and entropic contri- tor peptide complex was used and the system was pre- butions.22,23,25,26 This expanded view includes a wide pared as described for the LXR–RXR complex but with- range of allosteric events, spanning from the extremes of out addition of water. The minimized LXR complex was a solely enthalpic mechanism with structural rearrange- cooled to 10 K during 1 ns, until the overall root mean ments to an entropic mechanism with only changes in square fluctuation (RMSF) in atom positions was less protein dynamics. Due to the structural stability of the than 0.05 A˚ . In this cold structure the side chain of a NR LBD it is likely that an allosteric signaling within the selected residue was coupled to a thermal heat bath at domain has a high entropic contribution. 300 K, whereas the rest of the system was left uncoupled. The receptor complexes were positionally restrained by a harmonic potential on the backbone CA atoms (5 kcal 2 2 METHODS mol 1 A˚ 2) and on atoms with an accessible surface area larger than 10 A˚ 2. Restraints on surface atoms were intro- Simulations duced by Ota and Agard to avoid energy transmission to- All molecular dynamics simulations were performed ward the solvent.24 Here, we have excluded the water, with the CHARMM program27,28 using lookup tables to because it has a very limited effect on the overall results

296 PROTEINS LXR Allosteric Communication Pathway given the restraints on surface atoms and the short simu- and PROX1-1,2) interacts with E284 or A283, whereas lation time. Furthermore, the presence of water signifi- the 16 site (NCOA2-2,3 and NCOA6-2) only interacts cantly extends the time required for the initial cooling of with A283. Other specific interactions are observed to- the system. Restraints are used to maintain methodologi- ward the peptide 25, 17, or 19 positions. Residues cal reproducibility. ATD MD simulations with velocity involved in specific interactions are often long and polar Verlet dynamics were performed for 10 ps. The diffusion amino acids, thus, they have the potential to reach41 of heat was detected by root-mean-square deviation over a large protein surface area to find a potential (RMSD) time series of the amino acid side chains. Regu- hydrogen bond partner. This flexibility is reflected in the lar MD simulations were also performed for the ATD atomic RMSF analysis, where cofactor peptide residues system solvated in an 85 3 65 3 65 A˚ 3 TIP3P water box involved in specific binding show high positional fluctua- before minimization, otherwise the simulation protocol tions. However, in a very stable specific interaction (high was the same as described for the LXR ATD simulated interaction occupancy) as in PROX1-1 (K-4 and N-2) or complex. A 10 ns trajectory was used for covariance anal- NR0B2-1 (R-4), the interaction restricts the side chain ysis on the entire system. fluctuations. All analysis was performed on trajectories after re- Peptides NCOA2-1, 2, 3, NCOA6-1, and NR0B2-2 moval of overall translation/rotation of the protein com- show at least one peak (>1A˚ ) in the RMSF of the side plex, using coordinate snapshots every 2–5 ps. The initial chains at the N-terminal end of the peptide (Fig. 4). The equilibrated structure was used as reference structure low N-terminal RMSF values of peptide PROX1-1 and unless otherwise stated. A hydrogen bond interaction is NR0B2-1 are due to hydrogen bond restriction, but the considered when the hydrogen and the acceptor are low N-terminal fluctuations of NCOA6-2 and PROX1-2 within 2.4 A˚ without angular criteria.37 The hydrogen remains elusive. Possibly, the strong charge clamp inter- bond occupancy is calculated over the 10 ns trajectory action with E441 tightens the interaction and thereby and occupancies above 5% are considered. The accessible reduces the fluctuations at the N-terminal end. Further- surface area is calculated with a probe radius of 1.4 A˚ .38 more, the side chains of the C-terminal residues show larger RMSF values than the N-terminal residues, for all peptides studied. This trend is also observed in the back- RESULTS bone RMSF where residues at position 18 and 19 show values above 0.5 A˚ . Apart from these and the most C-ter- LXR cofactor interactions minal 25 residue, backbone RMSF is below 0.5 A˚ . Small- The LXR–RXR dimer with different cofactor peptides est backbone fluctuations are observed at the 11 back- showed high stability during the 10 ns simulations. No bone position, but also position 21, 12, and 13 seems major structural changes were observed neither at the to be positionally stable. At the center of the peptide, the receptors, peptides or ligands (data not shown). The side chain of residue 11 shows low-RMSF values for all charge clamp interaction39,40 between the LXR peptide simulated peptides as does the most central part of the backbone and LXR K273 and E441 is observed in all sim- peptide (position 24to15). Flexibility can be further ulations (Fig. 2). The strength of the charge clamp inter- decreased by an internal salt bridge as in NCOA2-2, 3 action is reflected in the high occupancy number of the and NR0B2-2, but comparing all simulated peptides the hydrogen bonds involved. E441 occupancy was >39% in lowest RMSF values at the central part are observed for most simulations, whereas K273 ranges between 7% and peptides NCOA6-1 and NR0B2-1. 45%. In general the charge clamp interactions are distrib- uted when several peptide amino acids are involved in Allosteric signaling the interaction and the hydrogen bond occupancy is then reduced for each individual interaction pair. Utilization of the ATD method is greatly facilitated by Nonspecific binding is also observed toward the pep- prior knowledge, or an educated guess, of which residues tide N-terminal part [peptide residues 25to11 (pep- in the proteins are involved in the allosteric pathways. tide numbering starts with 11 at the first leucine in the We aim to study the communication pathway between LXXLL motif)] involving either D371 or K291 of the re- the cofactor and the ligand, thus, a residue at either end ceptor. For peptide residues C-terminal of L11, specific of this pathway can be selected for heating. Furthermore, interactions are frequently observed (NCOA2-2,3, the study on the PDZ protein24 showed that allosteric NCOA6-2, and PROX1-1,2). These are formed with the signal transmission is likely to be transmitted through receptor R227, Q266, E284 and often with R283 nonpolar side chains. Binding of cofactors to NRs is (NCOA2-2,3, NCOA6-2, and PROX1-2). The peptide guided by a conserved a-helical cofactor core motif specific interactions differ due to the amino acid type at (LXXLL), where the leucines fold to the nonpolar recep- the site. In our simulations the amino acids at positions tor AF-2 surface. The conserved leucine amino acids in 12 and 16 are often observed in specific interaction the motif are often explained as necessary for the hydro- with the LXR receptor (Fig. 3). The 12 site (NCOA6-2 phobic interaction,40,42,43 but we also believe that it is

PROTEINS 297 S. Burendahl and L. Nilsson

Figure 2 Hydrogen bond interactions between cofactor peptide and LXR receptor. Peptide sequence of (a) NCOA2-1, (b) NCOA2-2, (c) NCOA2-3, (d) NCOA6-1, (e) NCOA6-2, (f) PROX1-1, (g) PROX1-2, (h) NR0B2-1, and (i) NR0B2-2 are given within boxes and residues in a-helical conformation are shown with grey background. Interactions to the peptide backbone are indicated with a line and to the peptide side chain with dashed line. Saltbridges within the peptide are shown as dashed line between sticks with ball. also possible that they might be involved in allosteric sig- study if the signal could be transmitted in the opposite naling. The first (L11) and the last leucines (L15) in direction, from the ligand to the cofactor peptide, we the motif were therefore selected as a starting point for also performed ATD with heating of the ligand. The sig- the ATD heating. The ATD results showed an interesting nal transmission from the ligand cause increased RMSD pattern (Figs. 5 and 6). Heating of L11 showed a clear fluctuation of A294, I295 and of residues adjacent to heat transmission to the LXR residues L290, A294, I442 these on the receptor helix 5 (Supporting Information and W443. Increased RMSD is also observed at K291, Fig. 2). RMSD peaks were also observed at W443, I442, I295, peptide L15, and ligand T-17, but here, the signal K291 and L11. Only minor RMSD fluctuations were shows up as an increased fluctuation rather than a dis- observed at L15 and L290. tinct peak (Supporting Information Fig. 1). Heating on Taken together these results from heating at the cofac- L15 resulted in an RMSD peak for L11 and L290. The tor peptide and at the ligand, indicate that transmission A294, I295, W443 and T-17 show an increased RMSD of a signal can be possible between these two regions. fluctuation, but with lower amplitude than observed Along the pathway, a subset of amino acids is repeatedly when heating at L11. Heating on both L11 and L15 observed to participate in the signal transmission, but caused RMSD peaks with high amplitude of L290, K291, some sites (e.g., K291) sometimes lack a signal when I442 and W443, whereas A294, I295 and the T-17 expected. We decided to heat on K291 as well. The K291 showed a moderately increased RMSD fluctuation. To heating caused an RMSD peak at L290, L11, and A294

298 PROTEINS LXR Allosteric Communication Pathway

ATD heating on K291 without the backbone restraints. This indeed resulted in an increased signal transmission to the ligand and the cofactor peptide, mainly through the h5 pathway. However, because the RMSD also increases for the entire system the signal becomes less distinct and more difficult to follow. The signal distribu- tion per residue, in ATD simulations (heated on K291) with and without backbone restraints, are shown in Fig- ure 7(b). These plots illustrate how the RMSD increases for both the backbone and side chains when no back- bone restraints are used but also show how the signal becomes less clear. To determine a significant communication pathway, ATD heating on several residues around the suggested pathway was performed. Particularly, residues in the pep- tide–receptor interface, at h4 and around the LBP were Figure 3 studied (data not shown). Heating of these sites induces LXR cofactor binding interactions. Receptor (cartoon and surface representation) charge clamp residues E441 and K273 (sticks) interact an increased temperature in adjacent residues and occa- with the NCOA6-2 cofactor peptide backbone (green carton) amide sionally toward more specific sites. However, none of the nitrogens and carboxyl oxygens (sticks). A specific interaction is made other ATD heated residues give rise to a signal transmis- between the peptide 12 and 16 residues and the LXR R283. [Color figure can be viewed in the online issue, which is available at sion along a reproducible directional pathway. Similarly, wileyonlinelibrary.com.] heating of a randomly selected residue not interacting with the suggested allosteric communication pathway, gives rise to increased movements in the adjacent resi- (Supporting Information Fig. 3). The fluctuations of the dues but without a direction of the energy transmission RMSD increased for I295, W443, L15, and T-17. From (data not shown). Such energy redistribution is charac- this it was clear that K291 can transmit the heat signal teristic of residues not involved in an allosteric commu- both to the cofactor peptide and to the ligand. Signal nication pathway24 and verifies a correct ATD setup. transmission to the ligand can take the pathway through The covariance correlation analysis on the LXR back- A294 and I295 in h5 or through I442 and W443 in h12, bone (Fig. 8) revealed several medium strong correlations and to the cofactor peptide it is likely to pass from L290 (0.5–0.75); covariance matrices for LXR Ca in all nine via L11toL15, because heating of L15 does not heat cofactor complexes (Supporting Information Fig. 5) were K291 directly. quite similar, and rather featureless even though the ATD, thus, indicates two possible signaling pathways amount of motion in the LXR was normal as judged between the cofactor peptide and the ligand, one through from the LXR RMSFs (Supporting Information Fig. 6). h5 and one through h12. To determine if the pathways Some of the correlated movements are observed between could be utilized individually, we performed the ATD secondary structure elements which are closely located in heating on the W443. The results showed RMSD peaks space and have large contact areas, such as h8 to h9, h7 at I442, T-17, L11 and increased RMSD frequency at to h10/11 and h9 to the h9/h10 loop. Other correlations A294 and I295 (Supporting Information Fig. 4). Thus, are observed even though the contact areas between the the signal would use both the h5 and the h12 as trans- secondary structure elements are smaller, as the h3 to mission pathways. The transmission of heat was sug- h12 and h5 to h7. Correlation was also observed between gested by Ota and Agard24 to pass between amino acid h3 and h6 which have no direct contacts in the LXR through hydrophobic contacts by the side chains. This structure. A negative correlation was also observed type of signaling was indeed observed between the cofac- between the loop region often described as the ‘‘h2-loop’’ tor peptide and the receptor (L11 and L290) and recep- and the h6/h7 loop as well as to the h8/h9/h11. tor h4 to h12 to the ligand (K291 to I442 and W443 to T-17) in our study, but we could also observe signaling likely to be transmitted through the backbone. This was DISCUSSION the case for the signaling within the cofactor peptide and LXR cofactor interactions from L290 all the way to I295. These amino acids have no side chain van der Waals (vdW) contacts which could Comparing the peptide–receptor interactions of the explain the transmission, so the signal would be expected different systems revealed a general pattern. In NCOA2- to take a different route. It is possible that the presence 1, NCOA6-1, and NR0B2-2 the charge clamp interaction of backbone restraints leads to an underestimation of plays a major role for the interaction. The interaction such a backbone signaling. We therefore performed a test involves several of the cofactor peptide residues and lasts

PROTEINS 299 S. Burendahl and L. Nilsson

Figure 4 Positional fluctuations of cofactor peptides (RMSF). Backbone (red line) and side chains (green line) RMSF of (a) NCOA2-1, (b) NCOA2-2, (c) NCOA2-3, (d) NCOA6-1, (e) NCOA6-2, (f) PROX1-1, (g) PROX1-2, (h) NR0B2-1, and (i) NR0B2-2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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100% for E441 and 13%/13%/7%/14% for K273, for the four peptides, respectively) and the receptor–peptide interaction is reinforced by several strong specific interac- tions. Specific interactions are observed from both multi- ple peptide sites and receptor amino acids and the inter- action occupancies are high (34%/54%/33%/69%). To- gether these findings suggest that a cofactor peptide–LXR interaction can take place through different interaction patterns: in the first group (peptides NCOA2-1,2, NCOA6-1, PROX1-2, and NR0B2-2), there is a strong charge clamp interaction and no or very weak specific additional interactions. The cofactor peptide residues in this group show distributed positional fluctuations along the entire polypeptide chain, except for the stable N-ter- minal region of PROX1-2. The second identified group

Figure 5 Amino acids involved in signal transmission according to the ATD method. Signaling between LXR, NCOA2-2 peptide (blue cartoon) and ligand T-17 (spheres) can be transmitted through the LXR backbone or through side chains. The amino acid side chains (yellow sticks) involved in signal transmission according to the ATD simulations are presented by their connected vdW surface. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] through a major part of the simulation time. In NCOA2- 2 and PROX1-2 the strong charge clamp interaction is supported by an additional specific interaction with R283 at low occupancy (9%). In NCOA2-3, NCOA6-2, PROX1-1, and NR0B2-1 the peptide–receptor interaction is more complex. Here, the charge clamp interaction strength varies (interaction occupancy 100%/89%/12%/

Figure 7 RMSD of LXR and peptide. (a) RMSD of backbone atoms in a nonheated ATD simulation at 100 ps (black), and with heating on amino acid K291 at times 1 ps (red) and 6 ps (green). (b) RMSD of side chain atoms in a nonheated ATD simulation at 100 ps (black) and Figure 6 with heating on amino acid K291 at times 1 ps (red) and 6 ps (green). Inter and intra molecular signaling in LXR detected by the ATD (c) RMSD of backbone atoms in ATD simulations with heating on method. Signal transmission is indicated by arrowed line between the amino acid K291 at 1 ps, with (black) and without (red) backbone residues (oval shape). When the arrowed line is complemented by a restraints. (d) RMSD of side chain atoms in ATD simulations with solid line, the signaling often passes through the polypeptide backbone heating on amino acid K291 at 1 ps, with (black) and without (red) otherwise transmission is taking place by side chain vdW contacts. ATD backbone restraints. [Color figure can be viewed in the online issue, heating has been performed on residues marked with a flash. which is available at wileyonlinelibrary.com.]

PROTEINS 301 S. Burendahl and L. Nilsson

tern, previously described for the liver receptor homo- logue-1 (LRH-1).44 The triangular interaction pattern can be involved in both the orientation of the cofactor peptide and affect the affinity through the specific R283 interaction (D372 in LRH-1). In LXR, the R283 interac- tion with cofactor peptide residue 12 is less frequently observed and a more distributed interaction pattern is revealed compared with the LRH-1 interactions. This might indicate that the LXR cofactors use specific inter- actions in the peptide to a lesser extent than LRH-1, and therefore an LXR cofactor would bind with less affinity in a more unspecific manner. The cofactor peptides used in this study can function as coactivators (NCOA2 and NCOA6) or corepressors (PROX1 and NR0B2). The interaction patterns indicate that the coactivator and corepressor peptides interact with the receptor in similar ways. Both nonspecific bind- Figure 8 ing, mainly through the charge clamp, and a more spe- Covariance correlation analysis of LXR backbone. LXR sequence is cific interaction through side chains was observed for shown as secondary structure elements. Correlation was observed both the coactivator and corepressors. In the RMSF anal- between secondary structure elements closely located in the receptor, ysis, there is a tendency that the coactivator side chains but also between h3 and h6 which have no direct contacts in the LXR structure. Possibly this correlation can be mediated by a bound ligand. toward the N-terminal region of the peptide have larger The analysis was based on a 10 ns simulation data if the solvated LXR fluctuations than the corepressors [considering the four with ligand T-17 and NCoA2-2 cofactor peptide. [Color figure can be first amino acids in each peptide, 9 out of 20 amino viewed in the online issue, which is available at wileyonlinelibrary.com.] acids in the coactivator set, Fig. 4(a–e), have RMSF > 1.0 A˚ , whereas in the corepressor set, Fig. 4(f–i), 2 out of also shows a strong charge clamp interaction, but the 16 amino acids have RMSF > 1.0 A˚ ]. Such small posi- cofactor–receptor interaction is reinforced by an addi- tional deviation in the peptide binding could alter the tional strong specific interaction as observed for peptides binding mode for the larger cofactor macromolecule, NCOA2-3 and NCOA6-2. Both peptides in this group causing effects on the biological response. A difference in show lower RMSF in the N-terminal end than in the binding mode can also be exploited for drug discovery C-terminal. Lastly, the PROX1-1 and NR0B2-1 peptides purposes, using the cofactor receptor interaction as drug have a very weak charge clamp interaction, but here, target. Possibly binding of a small molecule or structural other strong interactions play a major role. Interestingly, modification at the N-terminal interaction surface might it seems like the charge clamp residue E441 here interacts reduce the interactions with corepressors, leaving the in a specific manner to the peptide side chain. This looser coactivator interaction unaffected. Furthermore, it causes the exceptionally low-RMSF values of the peptides would be of interest to see how mutations in the cofactor in the N-terminal region. polypeptide sequence would influence receptor binding. Apart from the charge clamp residues, several other Our simulations also indicate that additional specific amino acids interact with the cofactor peptide. Many of interactions could be obtained with less positional fluctu- the receptor amino acids interact occasionally, but some ations. Therefore, it would be of interest to see how are observed more frequently such as R283. R283 is mutational studies of long polar residues to shorter polar located at a central position of the peptide close to the variants would modify the peptide–receptor interaction. peptide 12 residue but can with its long and flexible side chain reach residues from residue 22to16.41 In our Allosteric signaling peptide simulations, a hydrogen bond to residue 12 was only observed when a serine held the position (NCOA6-2 The ATD simulations revealed signal transmission and PROX1-2). In peptide NCOA2-2, the 12 histidine from the cofactor peptide to the ligand in the LXR LBP was found to hydrogen bond to R238 in the X-ray struc- (Figs. 5 and 6). The cofactor peptide leucine at the ture, but the interaction was not stable during the 10 ns N-terminal end of the LXXLL-motif (L11) was shown to MD simulation. In most of the simulated peptides, a be a key residue of the peptides in this pathway. From hydrogen bond interaction to the 12 site is not possible this site the signal was transmitted to LXR L290 and and the R283 folds in another direction. In the simula- K291 located on h4. After K291 the signal can take two tion of NR0B2-2, R283 forms a hydrogen bond to the routes, either pass over to h12 I442 and W443 through a backbone of the 23 proline. Together with the charge vdW contact of the side chain or continue to h5 through clamp residues R283 forms a triangular interaction pat- the backbone. The signal splitting at K291 can explain

302 PROTEINS LXR Allosteric Communication Pathway the occasional lack of signal at this position if the signal h5 and h5 which could be related to the allosteric com- takes the backbone route and therefore becomes more munication pathway observed in the ATD simulations difficult to detect with the ATD method. From W443 the where seen. The lack of such correlated signal can be due signal can further on continue to the ligand through to insufficient sampling time for analysis (10 ns), or a vdW interactions. W443 and H421 have been suggested communication mechanism separated from a correlated to play a major role in the activation mechanism in both movement. In a study of tRNA synthetase an allosteric LXRa30 and LXRb.12,45 In LXRb T-17 forms hydrogen pathway between the anticodon region and the aminoa- bonds only to H421 (H435 in LXRb), which interacts cetylase region was detected using cross-correlation anal- with W443 (W457) in an energetically favorable T-stack- ysis and a network analysis, protein structure graphs.47 ing.46 The H421–W443 interaction stabilizes h12 and the Protein structure graphs can identify residues connected AF-2 region in an active conformation. Mutation of to each other by noncovalent interactions. The shortest LXRb W443 to other aromatic amino acids does not identified protein structure graph pathway between the affect T-17 activity12 which it should do in LXRa, anticodon region and the aminoacetylase region includ- because W443 participates in hydrogen bonding to T-17. ing the cross-correlated residues was selected as the allo- However, for the ATD signaling it is possible that other steric communication pathway. Similarly, del Sol aromatic amino acids than tryptophan can mediate the et al.48,49 search efficient communication pathways by signal transmission to T-17, because the transmission is identifying linker residues between defined modules of not hydrogen bond dependent. the protein. The results from these studies reveal that Transmission through backbone of h5 can possibly intermolecular interactions provide the fastest way for transmit the signal to distal sites like M298 at the end of communication. Thus, signal transmission was found h5. Along h5 several possible interaction points with the between residues through vdW interactions, as observed ligand are present. Several of the residues identified in in ATD simulations as well. However, the signaling in the the ATD study have also been identified with the SCA tRNA synthetase study was suggested to strictly pass method,16 such as L290 and K291 on h4 and A294 on through noncovalent bonds, in contrast to studies done h5. This suggests that these residues have coevolved with the elastic network model of signal propagation in through evolution and also are energetically coupled. On proteins.50 In an elastic network study of the phospholi- the other hand, some residues were identified by the pase A, secondary structure elements appeared to be effi- SCA method but not by the ATD method. This can be cient mediators of communication. Furthermore, the explained due to the fact that the SCA method identifies speed of the signal transmission from one residue to all coupled residues in the protein complex, whereas the another was highest within a-helical residues followed by ATD method focus on a more local coupling. Moreover, b-strands and then loops although other studies reveal the ATD simulations reflect communication in the ago- similar transmission speed for all secondary structure ele- nist T-17-LXR setup. It is possible that other bound ments.51 Nonequilibrium MD simulations by Nguyen ligands would induce a somewhat different communica- et al. also show that energy transfer occurs mainly tion pathway due to the unique molecular interactions of through the peptide backbone and that such transmission each receptor–ligand pair. It is also likely that a disrup- does not depend strongly on the MD force field.52 Thus, tion of the allosteric communication pathway found in these studies indicate that signaling through covalent the LXR-T-17 system might alter the pathway. The effec- bonds in the backbone plays a major role for signal tiveness of communication through a modified pathway propagation. Our ATD study of the LXR showed that sig- might be changed compared with the LXR-T-17 pathway. naling was likely to utilize pathways through both nonco- As a consequence of unique communication pathways, a valent contacts and covalent bonds. The combinatorial ligand specific transcriptional response can be achieved. signal transmission is consistent with the thermodynami- Our results indicate that both the extent of the energetic cal view on protein allostery.22,25,26 coupling and its transmission pathway can be modified According to the thermodynamic view, both the and tune the receptor response between activation and enthalpic and entropic components of the free energy inhibition. may be involved in allostery and complement each other. Allosteric signaling has been investigated with various As suggested by Tsai et al.,26 allosteric communication computational methods. Frequently, cross-correlation over shorter distances might have a higher entropic con- analysis of MD simulations has been used to map out tribution which is reflected by backbone entropic changes connected residues located at distal sites. Covariance and side chain rotations. For longer distance communica- analysis of the LXR receptor with bound ligand (Fig. 8), tion, larger structural changes and a more enthalpic con- reveal correlated movement between secondary structure tribution would be required. Our study of the allosteric elements located next to each other and therefore such communication between the cofactor peptide and the correlation is natural. The correlation between h3 and h6 ligand fits well into the short distance description with can possibly be mediated through the ligand in the LBP. both entropic changes in the h5 backbone and side chain However, no secondary structure correlation between h4, rotations along the obtained pathway.

PROTEINS 303 S. Burendahl and L. Nilsson

The nature of allosteric signal transmission and how tial cooling of the system, correction for rotational and energy flows through a protein are highly interesting translational movements in the trajectory before analysis research areas. Transport of vibrational energy has turned and short simulation time to avoid heating of the system. out to be specific and anisotropic.24,52–54 Our results The results from ATD simulations are best analyzed with indicate that changes of dynamic properties like vibra- both positional changes (RMSD) and fluctuation around tions of bonds and angles might explain the physical a position (RMSF), due to different behavior of amino mechanism of allosteric signal transmission. However, acids in response to a heat pulse. When receiving an the ATD simulations were performed at a temperature ATD signal residues with small side chain tend to fluctu- close to 0 K and the temperature dependence and other ate rather than flip, whereas amino acids with large side environmental conditions have to be thoroughly investi- chains show a more distinct conformational change. Fur- gated before one can draw general conclusions on the thermore, the use of positional restraints for the back- exact mechanism.51,52,55–58 Studies spanning from bio- bone and accessible surface residues is a limitation of the chemical experiments22,23,25 to MD calcula- method, and a methodological development to avoid tions51,55,56,58 confirm the importance of vibrational these positional restraints would significantly improve movements. Vibrations are one of the fundamental the method impact and accuracy of the results. With the movements in dynamics of molecules. Vibrational move- correct setup and treatment of data, the ATD method ments are also highly sensitive to environmental factors can be used to gain new insights into the communication like temperature, solvent, and molecular interactions. pathways within and between molecules. Identification of Thus, changing vibrational properties can be used as an the communication pathway might provide information effective signal on the molecular level. Vibrational signals on which residues that can be connected to a specific bi- can be transferred through a vdW interaction surface ological response. It is for example possible that an ago- between side chains but also through the backbone of nist makes use of a somewhat different communication the polypeptide chain, as shown in our ATD simulations. pathway than an antagonist. Thus, a more complete Changes in vibrations are difficult to observe in a MD understanding of allosteric communication and its path- simulation, due the large number of naturally existing ways might be valuable in a drug discovery process of vibrations within biomolecules. Although difficult to the target protein. Specific information of key interaction treat, noise might improve efficient signaling by enhanc- residues in allosteric signaling can be used for a struc- ing sigmoidal responses and switch-like behavior.59,60 tural based drug design. Moreover, experimental studies Covariance analysis of a MD trajectory measures the cor- of the peptide–receptor interaction would complement related movement of the molecules dynamics. The covar- theoretical ATD simulations. According to our simula- iance analysis of the LXR backbone indicated that regions tions of cofactor peptides–receptor and the ATD simula- around the LBP have a correlated movement. Some of tions, mutational studies of the peptide 11 residue these correlations might be induced due to interactions would be very interesting. The 11 residue is important between the secondary structure elements but others are both for the peptide–receptor interaction and the alloste- likely to communicate through other pathways, possibly ric communication within the domain, and therefore a the ligand. Such might be the case for the correlation key interaction for the receptors transcriptional response. observed between h3 and h6. The covariance analysis of the backbone provides information of which secondary structure elements that are likely to participate in an allo- CONCLUSIONS steric communication. Recent studies on the PDZ2 domain identify individ- Understanding communication within a biomolecule is ual correlated residues from a interaction energy correla- essential for the understanding of protein function. Inter- tion matrix.53 Two continuous interaction pathways were actions with cofactor peptides reveal the strength of the found, where one is also described by previous stud- charge clamp interaction for LXR. Additional specific ies.15,24,54 The second identified pathway might occur interactions were frequently found to the more C-terminal due to the extended number of pertubated residues. end of the cofactor peptides. This region also shows some- However, covariance analysis of MD trajectories gives what larger positional fluctuations, indicating that this fewer details on the mechanism of the signal transmis- region might be a good area for an LXR targeting drug sion than ATD results. Modified MD methods like the design. A possible communication pathway between the ATD method can with advantage provide additional in- coactivator peptide, LXR and ligand, found in the ATD formation on properties of biomolecules. Nonetheless, simulations, reveals a bidirectional communication one should always treat information from modified MD between the coactivator peptide and ligand. The first leu- methods with care. The ATD results are due to compari- cine in the coactivator recognition motif (LXXLL) was sons of small differences, where the RMSD peak of a identified as a key signaling residue. Due to the high con- transmitted signal mostly is <1A˚ . Thus, the ATD servation of the LXXLL-motif within the NR cofactors, it method is sensitive to noise and requires substantial ini- is likely that the found pathway can be used in other NR

304 PROTEINS LXR Allosteric Communication Pathway complexes. Moreover, allosteric signaling can also contrib- 10. Quinet EM, Savio DA, Halpern AR, Chen L, Schuster GU, Gustafs- ute to biological specificity, because the communication is son JA, Basso MD, Nambi P. (LXR)-beta regulation highly dependent on molecular interactions between the in LXR alpha-deficient mice: implications for therapeutic targeting. Mol Pharmacol 2006;70:1340–1349. receptor and ligand. The obtained pathway revealed signal 11. Farnegardh M, Bonn T, Sun S, Ljunggren J, Ahola H, Wilhelmsson transmission both through the backbone and through A, Gustafsson JA, Carlquist M. The three-dimensional structure of vdW contacts between side chains. This dual transmission the liver X receptor beta reveals a flexible ligand-binding pocket manner suggests the possibility of signaling at multiple lev- that can accommodate fundamentally different ligands. J Biol Chem els, where backbone signaling can be obtained in a 2003;278:38821–38828. 12. Williams S, Bledsoe RK, Collins J, Boggs S, Lambert MH, Miller sequence-independent fashion, whereas side chain signal- AB, Moore J, McKee DD, Moore L, Nichols J, Parks D, Watson M, ing is sequence dependent. Consequently, allosteric signal- Wisely B, Willson TM. X-ray crystal structure of the liver X recep- ing can be modified by sequence alteration. tor beta ligand binding domain: regulation by a histidine-trypto- Some LXR coactivator and corepressors competitively phan switch. J Biol Chem 2003;278:27138–27143. bind the LXR AF2 region and both bind the receptor with 13. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, Mangels- dorf DJ. LXR, a nuclear receptor that defines a distinct retinoid the LXXLL motif. Thus, the allosteric communication response pathway. Genes Dev 1995;9:1033–1045. pathway found for a coactivator can possibly utilized by a 14. Perutz MF. Myoglobin and haemoglobin: role of distal residues in LXR corepressor too. The interactions pattern between the reactions with haem ligands. Trends Biochem Sci 1989;14:42–44. receptor and the coactivator/corepressor peptides studied 15. Lockless SW, Ranganathan R. Evolutionarily conserved pathways of showed common features according to our MD simula- energetic connectivity in protein families. Science 1999;286: 295–299. tions. For most of the cofactors the charge clamp interac- 16. Shulman AI, Larson C, Mangelsdorf DJ, Ranganathan R. Structural tion plays a major role, but for the PROX1-1 peptide inter- determinants of allosteric ligand activation in RXR heterodimers. actions to the 12 and 17 side chain were more important Cell 2004;116:417–429. than the charge clamp. Several of the peptides showed a 17. Young MA, Gonfloni S, Superti-Furga G, Roux B, Kuriyan J. specific interaction toward the C-terminal side of the pep- Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphoryl- tide. Additional specificity for the LXR cofactors are prob- ation. Cell 2001;105:115–126. ably found in interactions to residues outside the central 18. Fidelak J, Ferrer S, Oberlin M, Moras D, Dejaegere A, Stote RH. recognition motif as observed for the full length cofactor Dynamic correlation networks in human peroxisome proliferator- DAX-1 interaction to LRH-1,61 binding to the ligand-in- activated receptor-gamma nuclear receptor protein. Eur Biophys J dependent activation function as observed for NR0B262 or 2010;39:1503–1512. 1 19. Boehr DD, McElheny D, Dyson HJ, Wright PE. Millisecond time- in cofactor concentration levels in the cell. scale fluctuations in dihydrofolate reductase are exquisitely sensitive to the bound ligands. 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306 PROTEINS