Receptor residues contacting oxygen-containing moieties

672 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679 tera and Lepidoptera e.g., Henrich et al., 1990; Oro et al., 1990; Shea et al., 1990; Tzertzinis et al., 1994; Kapit- skaya et al., 1996; Jindra et al., 1997; Perera et al., 1998; Vogtli et al., 1999. The sequence identity between these USPs versus the vertebrate and invertebrate RXRs is in the range of 40–50, while that between invertebrate RXRs and vertebrate RXRs is around 70. However, these statistics in and of themselves do not provide a basis for inferring whether USPs bind ligands and what those ligands might be. For example, vertebrate RAR and RXR both bind the same ligand 9-cis RA, yet the percent identity between them is only around 27 Mangelsdorf et al., 1990. The relevant indicators are whether the residues that have been retained identically or with conservative substitution are those residues necessary to preserve the secondary structures for basic architecture of the ligand-binding domain, and those necessary to preserve a functional ligand-binding pocket within the ligand-binding domain. Thus, in the analyses below, we do not confer importance to a residue in USP merely because it is conserved in USPs, but rather also because of its location in USP relative to the functional location of ligand- or co-activator-associated residues conserved in other nuclear receptors.

2. Secondary and tertiary structure

The crystal structure for unliganded human RXR α hRXR α has been reported, including the residues part- icipating in the various secondary structures Bourguet et al., 1995. It is possible to gauge the accuracy of algorithms for prediction of secondary structure by com- paring the consensus predictions of several methods for hRXR α against the secondary structure arrangement actually observed in crystallized hRXR α . Using the vari- ous secondary structure prediction alogithms available at the http:pbil.ibcp.frNPSA web site, we performed such an analysis. Fig. 1 shows the consensus locations of pre- dicted secondary structures for hRXR α vs those actually observed in the crystal structure, for the regions of the ligand-binding domain that contain corresponding resi- dues known to line the ligand-binding pocket of hRAR γ . In general, the analyses reasonably predict for hRXR α the locations of α -helices. We then applied those analyti- cal methods to Drosophila melanogaster USP dmUSP. The predictions of α -helical secondary structure for dmUSP generally parallel the predictions for hRXR α Fig. 1. These data on the predicted arrangement of sec- ondary structures in dmUSP support the hypothesis that the general tertiary architecture of the ligand-binding domain LBD of dmUSP is similar to that of the LBD for hRXR α . The remaining analyses below are rested on this hypothesis that the general tertiary architecture of secondary structures of the LBD of dmUSP is similar to that of the LBD for hRXR α .

3. Receptor residues contacting oxygen-containing moieties

The crystal structure of the ligand-binding domain has been reported for apo-hRXR α , holo-hRAR γ , holo-rat thyroid hormone receptor rTR β 1, holo-human estrogen receptor hER, and both apo- and holo-human pro- gesterone receptor hPPAR γ Bourguet et al., 1995; Renaud et al., 1995; Wagner et al., 1995; Brzozowski et al., 1997; Nolte et al., 1998; Shiau et al., 1998; Tanen- baum et al., 1998; Uppenberg et al., 1998; Williams and Sigler, 1998; Xu et al., 1999. The ligand-binding pock- ets of these LBDs are lined primarily with hydrophobic amino acid side chains Fig. 1. Those residues in the pocket that are charged are in most cases those residues that hydrogen bond with hydroxyl, keto or carboxyl groups of the respective ligands Fig. 2. In every case examined thus far of a crystallized or modelled, for hRXR α nuclear receptor complexed with an endogen- ous ligand containing a ‘terminal’ hydroxyl, keto or car- boxyl group at its ‘leading end’, that group is hydrogen bonded to two residues which each are placed on differ- ent secondary structures; either one on α -helix 3 and the other on α -helix 5, or one on α -helix 5 and the other on a β -turn S1–S2 Figs. 1 and 2. Thus far, the hydrogen bonds of the hydroxyl-, keto- and carboxyl-containing moieties always include at least a basic residue arginine or histidine on either α -helix 3 or 5, and a nonbasic, but hydrogen-bonding residue glutamine, glutamic acid or serine on either the other helix or on the β -turn Fig. 2. hPPAR γ , which has some additional secondary struc- tures and a tertiary arrangement most different from the other nuclear receptors, also binds the carboxyl group of its ligand through histidine on α -helix 5, but is excep- tional in that a histidine on α -helix 11 also hydrogen bonds with that same carboxylate group. On the basis of the above pattern of the nature and locations of hydro- gen-bonding residues in a variety of nuclear receptors, we make the following hypothesis: if USP binds to an endogenous ligand containing an oxygen-substituted end-group, that binding will be by hydrogen-bond of that end-group through at least arginine or histidine contacts at one of either α -helices 3 or 5, and perhaps through a second contact on the other respective α -helix 3 or 5 or on the β -turn. In hRXR α , the apparent closest relative of dmUSP that has been crystallized, the modelled structure of 9- cis RA in the RXR ligand-binding pocket places I268 and Q275 on α -helix 3 as straddling on each side of the ligand, near the backbone double bond positioned between the α and β carbons respective to the carboxyl group C14 and C15, Fig. 2. Those two residues are conserved in higher insect USPs as an invariant L and the correspondng invariant Q, respectively. Closer examination of the the residues on α -helices 3 and 5 and the β -turn that contact the oxygen-substituted 673 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679 674 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679 Fig. 1. Conservation of primary, secondary and tertiary structure of portions of ligand-binding domain of vertebrate nuclear receptors in comparison with primary and inferred secondary and tertiary structure of portions of ligand-binding domain of ultraspiracle USP proteins from higher insects. For the vertebrate receptors, the α -helices barrels or β -sheet block arrows contain the primary sequence shown by crys- tallography to constitute that secondary structure in the respective receptor. Open rectangles around hRXR α and around the sequence for dmUSP show the consensus of secondary structure predictions. Resi- dues coded in orange are present in the ligand-binding pocket of the crystallized receptor. Residues coded in dark brown touch oxygen-con- taining moieties on the ligand in the ligand-receptor cocrystal. Blue bars connecting residues in two receptors are positions in which differ- ences in hydrophobic side chains discriminate between ligands of the two receptors. Insect USP and dmE75A sequences are shown in align- ment below the vertebrate receptors. Light brown coded residues denote highly conserved insect sequences at selected positions. Purple coded dmE75 residues denote positions in which the dmE75 residue is a non-conservative change from the conserved series at the respect- ive USP position. Dark brown underlining for α -helices 3, 1011 and the β -turn indicate narrower regions in vertebrate receptors that contact oxygen-containing moieties on the respective ligands. Primary sequences and alignments for vertebrate receptors are from Wurtz et al. 1996. Sources for secondary and tertiary structure designations and receptor acronyms are as follows: hPR = human progesterone recep- tor Williams and Sigler, 1998; hER = human estrogen receptor Brzozowski et al., 1997; hRAR γ= human retinoic acid receptor γ Renaud et al., 1995; hRXR α= human retinoid X receptor α Bourguet et al., 1995; rTR β 1 = rat thryoid hormone receptor β 1; dm = Drosophila melanogaster Oro et al., 1990; aa = Aedes aegypti Kapitskaya et al., 1996; ct = Chironomus tetans Vogtli et al., 1999; bm = Bombyx mori Tzertzinis et al., 1994; cf = Choristoneura fumiferana Perera et al., 1998; ms = Manduca sexta Jindra et al., 1997. leading end of ligands of a variety of crystalized ligand- receptor complexes leads to additional hypotheses on candidate residues in dmUSP that may also contact an oxygen-substituted endogenous ligand. The residues on α -helix 3 reported to hydrogen-bond with the hydroxyl-, keto-, or carboxyl moiety of the respective ligands are all in positions within 5 amino acids of each other on helix 3 Fig. 1. In this immediate area on hRXR α are also the I268 and Q275 that are modelled as just distal to the leading carboxyl group. The only invariant resi- dues in that region for the USPs are those in the motif LXXXXXKQ, where L and Q correspond to I268 and Q275 of hRXR α . Further, on α -helix 5, hPR, hER, hRAR γ and hRXR α all possess a conserved arginine that participates in hydrogen-bonding to the hydroxl-, keto or carboxyl- moiety of their ligand. However, in several of the USPs, this residue is neither arginine nor another basic residue. Based on the nature and locations of con- served residues in USP α -helix 3 relative to the corre- sponding placement of ligand-associated residues in other crystallized receptors, we hypothesize that should higher insect USPs bind an endogenous ligand with ter- minal oxygen-substitution, then either the conserved K or Q on helix 3 participates in hydrogen-bonding to that moiety. More problemmatic is conjecturing which residues are candidates for a second hydrogen bond with the oxygen- containing moiety of a putative USP ligand. The short region between S1 and S2 of hRAR γ and rTR β 1 contains in those receptors the serine residue making the second hydrogen bond to the leading-end oxygen-containing moiety. It is likely of significance that this short region has been substituted for a larger and more variable region in the higher insect USPs Fig. 1. This region in each USP apparently contains an invariant aspartic acid, in addition to at least one serine. Also relevant to this analysis is comparison of the status of these hypothesized ligand-associated residues of USP with the corresponding residues in dmE75A. This latter regulator has also been hypothesized to be an orphan receptor, or, at least, there have been no reports of it binding to juvenile hormones or JH-like molecules Thummel, 1995; Buszczak and Segraves, 1998. dmE75A does not conserve the invariant Q on α -helix 3 of USP which is instead V417, the invariant K on α -helix 3 is instead H408, and the invariant IL on α -helix 3 is instead F410. In addition, two other resi- dues on that α -helix 3 that substantively participate in creating the hydrophobic environment of the ligand- binding pocket in each of the crystallized vertebrate receptors have been changed to charged E and R resi- dues at the corresponding positions in α -helix 3 of dmE75A E407 and R413. On α -helix 5, dmE75A has also replaced an otherwise invariantly hydrophobic USP residue IL with a charged R452. Likewise, a hydro- phobic residue otherwise invariantly conserved on helix 7 in both RXRs and USP, and which contributes to the hydrophobicity of the pocket in several receptors, is instead a polar threonine residue T491 in dmE75A. One current hypotheses is that E75A and USP are orphan receptors that do not have a ligand. Another is that either may have a ligand that is as yet unknown. There has been discussion in the literature of whether the differences that exist in identity of residues between the USPs and RXRs in the ligand-binding domain reflect that USP does not have a ligand-binding func- tion, i.e. there would then be a loss of selection for maintenance in USP of those RXR-particular residues Kapitskaya et al., 1996; Hayward et al., 1999. Under the hypothesis that USP and E75A are orphan receptors with no ligand, neither E75A nor USP would be under selection pressure to maintain a ligand-binding pocket architecture that is favorable for binding hydrophobic, end-oxygenated ligands. However, the analyses here on comparison of the differences between dmE75A and RXRsUSPs for conservation of ligand-associated resi- dues in RXR α -helices 3 and 5 raises the important con- sideration of why USP does not show similar encroach- ment of charged residues in its ligand-binding pocket as does dmE75A, if USP is not under selection pressure to maintain a hydrophobic ligand-binding pocket to bind hydrophobic ligands? This consideration does not 675 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679 Fig. 2. Summary of specific hydrogen bonding contacts between specific ligand-binding pocket residues and oxygen-containing motifs of the respective ligands, as shown by co-crystallography. The residue of a particular color moiety of the same color for each receptor-ligand combination shown. Also provided are the hydrogen-bonding distances from the indicated residue to the respective site on the ligand, or to a water molecule that in turn h-bonds with the oxygen-containing moiety. EPA = eicosapentaenoic acid; AT-RA = all trans retinoic acid; DIMIT = 3,5-dimethyl-3 9- isopropylthryonine. Each specific oxygen-containing moiety shown is a part of the indicated ligand and not an extra moiety added to it. All other abbreviations as in Fig. 1. imply that E75A does not have a ligand, but rather inquires as to why the USPs, but not E75A, have been selected to conserve residues that in RXR provide a hydrophobic ligand-binding environment. The end of the ligand-binding pocket that is near the ligand entrance point shows more divergence among receptors. However, a narrow region on α -helix 11 is the location of residues that in some crystallized recep- tors make additional hydrogen-bonds, again with other hydroxyl or keto moieties that are also present on the opposite end of the respective ligand e.g., progesterone and thryoid hormone, Figs. 1 and 2. In both hER and rTRb1, it is a basic histidine that makes the respective hydrogen-bond Fig. 2. In the same narrow respective region of dmUSP is an invariant K472 and an invariant H476, neither of which is conserved in dmE75A although the change from histidine in dmE75A by K578 is conservative. Based on these available data, we hypothesize that if USP does bind an endogenous ter- penoid ligand through a hydrogen bond to an oxygen- containing moiety on the ligand’s distal end, then either the conserved lysine or conserved histidine participates in that bond. We also note that the insect juvenile hor- mones possess an oxygen-substitution epoxide group at its end opposite from its carboxyl ester group, and that we did not observe methyl farnesoate, that is miss- ing the epoxide, to bind to dmUSP Jones and Sharp, 1997. 676 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679

4. Conservation of residues participating in ligand- induced coactivator-binding