Conservation of residues participating in ligand- induced coactivator-binding

676 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679

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

There has been intense investigation of the nature of transcriptional coactivators that are recruited to bind to the new conformations adopted by vertebrate receptors upon binding with their respective ligand Glass et al., 1997; McKenna et al., 1999. Thus far, three different transcriptional coregulators that are recruited to RXR of the heterodimers PPARRXR, RARRXR and BORRXR are only recruited to the RXR partner after RXR binds its ligand 9-cis RA Lee and Wei, 1999; Monden et al., 1999; Wiebel et al., 1999. Recently, coactivator binding to the ecdysone receptor has been demonstrated Tsai et al., 1999. Several vertebrate receptors have been cocrystalized in complexation with such a recruited coactivator, giving precise identification of receptor residues that constitute the surface that binds the coactivator. Part of this surface is contributed by resi- dues in α -helix 12, which upon receptor binding of agonist ligand moves to contact α -helices 3 and 4. Two differently functioning parts of this combined, coacti- vator-binding surface have been identified. One is a hydrophobic groove in which the floor is lined with hydrophobic residues and the rim with polar residues. The other is a ‘charge clamp’ where a basic residue con- tributed by α -helix 3 and an acidic residue contributed by α -helix 12 assist to hold in place the recruited co- activator that has inserted its LXXLL motif into the hydrophobic groove Darimont et al., 1998; Nolte et al., 1998; Shiau et al., 1998. As shown in Fig. 3, higher insect USPs have invariantly conserved on α -helix 3 the presence of a basic arginine at the same location as is conserved the charge clamp lysine residue in the vertebrate receptors. dmE75A instead has a glycine at this position E75A G454. The insect USPs have also conserved a number of the corresponding hydrophobic residues on α -helices 3 and 4 that in the vertebrate receptors contribute to the Fig. 3. Conservation in insect USPs of α -helix 3 and 4 residues contributing in vertebrate receptors to the hydrophobic groove for ligand-induced coactivator binding. Dark brown coded residues are those observed in co-crystals to form part of the hydrophobic groove. Light brown are those residues in other vertebrate receptors and USPs that conserve the residues forming the hydrophobic groove. Dark blue is the basic lysine residue that constitutes one side of the “charge clamp” holding the co-activator in place as seen in cocrystals, while light blue coding shows the conservation of a basic residue at this location in other receptors. Pink-coded residues are not conserved. Abbreviations as in Fig. 1. Sources for sites of co- activator binding deduced by crystallography are: rTR β 1, Darimont et al. 1998; hPPAR γ , Nolte et al. 1998; hER, Shiau et al. 1998. formation of the hydrophobic groove. Crystallography has also shown that a glutamine invariantly conserved in the vertebrate RXRs and the insect USPs is an integral part of the framework for the coactivator binding region Shiau et al., 1998. That glutamine is not conserved in dmE75A K467. Helix 12, the other major contributing secondary structure to the hydrophobic groove, also shows the rel- evant conservation of residues in higher insect USPs. As shown in Fig. 4, an acidic residue is invariantly con- served in the vertebrate receptors that constitutes the other side of the ‘charge clamp’. Also conserved in the vertebrate receptors are several locations of hydrophobic residues, at least one position of which contributes a hydrophobic residue to the floor of the hydrophobic groove mentioned above. In the vertebrate receptors, α - helix 12 is amphipathic. Its residues are organized in such a way that when ligand binds it induces α -helix 12 to move to contact α -helices 3 and 4. This movement thereby closes the ligand-binding pocket, and the con- served residues that contribute to the charge clamp and the floor of the hydrophobic groove are on one face of the helix. Residues constituting the hydrophobic lid over the ligand-binding pocket are on the opposite face Fig. 4. The organization of residues in α -helix 12 of dmUSP also appears to follow an amphipathic pattern, conserv- ing a hydrophobic residue at the position for a hydro- phobic groove residue in vertebrate receptors, conserving the acidic residue that constitutes part of the charge clamp in vertebrate receptors, and conserving hydro- phobic residues on the opposite face that would puta- tively face into the ligand-binding pocket. In fact, the presence of an acidic residue at the charge clamp pos- ition is invariantly conserved in all higher insect USPs. Those USPs also all further conserve hydrophobic resi- dues at the position contributing to the hydrophobic groove in the vertebrate receptors dmE75A instead has a polar serine at that position S506. On the basis of these conservations in α -helices 3, 4 and 12 of ligandco- 677 G. Jones, D. Jones Insect Biochemistry and Molecular Biology 30 2000 671–679 Fig. 4. Structural analysis of AF-2 region of α -helix 12 in vertebrate and invertebrate nuclear receptors. Dark brown coded hydrophobic residues with trough symbol were observed to contribute to the hydrophobic groove that binds co-crystallized co-activators, while light brown coded residues are those conserved hydrophobic residues at the corresponding position in the indicated other receptors. Dark blue, encircled acidic residues are those observed to form part of charge clamp with the co-crystallized coactivator, while light blue, coded residues are those conserved acidic residues at the corresponding position in the other indicated receptors. Dark green hydrophobic residues are those that face into the hydrophobic ligand- binding pocket in the ligand-receptor cocrystal, while light green residues are those conserved hydrophobic residues at the corresponding position. Pink encoded residues are not conserved. Shown below the alignment is a helical wheel representation of the amphipathic α -helix observed for this region in crystalized holo-hPPAR γ and holo-hER, and apo-hRXR α , and a modeled representation of the potential similar structure of the corresponding region in dmUSP. Colors on different segments of the wheel have no structural significance and are included solely to aid in visualization of the direction of the spiral. For dmUSP, the green arc offers a possible face of the amphipathic helix that would face into the hydrophobic pocket in the putative ligand-induced conformation. The last two rows of residues shown are residues that vary between receptors and receptor isoforms that appear to contribute to receptor-specific binding responses to 160 coactivator family proteins. Sources for residues contributing to the site of co-activator binding, as deduced by crystallography are: rTR β 1, Darimont et al. 1998; hPPAR γ , Nolte et al. 1998; hER, Shiau et al. 1998. Abbreviations are as in Fig. 1. activator-associated residues, we hypothesize that if insect USPs bind an endogenous ligand, and then change conformation Jones and Sharp, 1997, part of that con- formational change is movement of α -helix 12 into a position that forms with helices 3 and 4 a hydrophobic groove constituting a co-activator binding site, complete with charge clamp.

5. Additional structural and functional considerations