The crystal structures reported here reveal how AR binds coactivator motifs with bulky aromatic hydrophobic groups and permit construction of a profile of the AR coregulator interface (see ). In some ways, this interface resembles those of other nuclear receptors: it is an L-shaped hydrophobic cleft comprised of three distinct subsites that bind hydrophobic groups at the +1, +4, and +5 positions in cognate peptides. Moreover, the so-called charge clamp residues (Lys720 and Glu897) bracket the cleft. Nonetheless, the AR coregulator recognition site is unique in that it rearranges upon motif binding to form a long, deep, and narrow groove that accommodates aromatic residues at the +1 and +5 positions (). Sequence alignments of AR with other NRs suggest that a unique combination of substitutions at Val730, Met734, and Ile737 combine to permit the formation of a smoother, flatter interaction surface that displays a higher complimentarily to aromatic substituents than to branched aliphatic (see ). Of these, methionine, the only unbranched hydrophobic amino acid and the most accommodating, at a key position between the +1 and +5 sites, allows the AR AF-2 interface to vary the size and shape of its pockets to associate with a more diverse set of coregulators. GR also contains a methionine residue at this position, raising the possibility that it may also employ induced fit to broaden motif recognition. While naturally occurring mutations in AR have yet to be observed at Met734, it is interesting to note that mutations at Val730 and Ile737 have been reported in patients with prostate cancer and androgen insensitivity, respectively (Newmark et al. 1992
; Quigley et al. 1995
; Gottlieb et al. 1998
Surface Complimentarity of Hydrophobic Motifs in the AR, ERα, and GR AF-2 Clefts
The same characteristics that make the AR AF-2 ideal for binding of longer, aromatic side chains also make it less well suited for binding of shorter, branched side chains. Although changes in the position of Met734 widen the groove towards the +5 subsite to permit binding of leucine residues, the gross features of the groove remain largely the same (see B). As a result, the +1 and +5 leucines bind in a smooth, elongated groove and interactions between the +1 and +5 residues on the face of the peptide helix, or with a hydrophobic “bump” present in other receptors caused by a isoleucine to leucine substitution between the +1 and +5 subsites, are absent. Thus, a smaller proportion of the available surface area is available for van der Waals interactions.
Unlike the conserved interaction modes of aromatic residues with the +1 and +5 sites, binding interactions at the +4 site are variable and characterized by nonspecific interactions. This finding agrees with the relatively high conservation of residues at the +1 and +5 positions of AR-interacting motifs and suggests that these residues drive peptide interaction with the LBD, whereas the +4 site is less critical. Indeed, the +4 pocket is shallow, surface exposed, and relatively featureless, explaining the assortment of residues selected at the +4 position. It is likely that any hydrophobic residue that does not clash with surrounding residues would be suitable at this subsite.
While peptide motif recognition is governed by hydrophobic interactions, polar interactions from backbone atoms and residues outside the core motif also contribute. With the exception of FxxFF, motifs containing phenylalanines at the +1 and +5 positions present canonical main chain interactions with both charge clamp residues, Lys720 and Glu897. This finding stands in contrast to predictions of previous studies (Alen et al. 1999
; He et al. 1999
; Slagsvold et al. 2000
; He and Wilson 2003
), which concluded that Lys720 was dispensable for FxxLF binding and that Glu897 was required for binding to FxxLF and LxxLL motifs. Lys720 comprises a significant portion of the +5 subsite, making important van der Waals interactions with the Phe+5 benzyl group in addition to hydrogen bonds to the motif backbone. These results suggest that Lys720 is required for binding of FxxLF motifs. However, it may be that enough binding energy is provided by the other residues of the +5 subsite (i.e., Met734), as well as by the other subsites themselves, such that removal of Lys720 would have little effect on binding. Observations that Lys720 plays a greater role in LxxLL motif binding are likely due to the fact that there is less surface area contributing to van der Waals contacts in LxxLL motifs. Disrupting binding contributions from Lys720 would thus have a more detrimental effect on binding.
On the other hand, Glu897 interacts with the FxxLF peptide backbone, but is disengaged from the LxxLL peptide backbone. One possible explanation for the apparent requirement for Glu897 in LxxLL binding is that it might interact with residues outside of the core motif. The corresponding glutamate of GR, Glu 755, forms hydrogen bonds with the −3 asparagine of TIF2 NR box 3 (Bledsoe et al. 2002
), and Glu897 of AR participates in noncanonical interactions with the hydroxyl group of a Ser−2 residue that was selected in all of our tryptophan-containing peptides. This is especially intriguing given that the only WxxLF motif known in vivo, located in the AR NTD, also possesses a Ser−2 residue. WxxLF also makes backbone interactions with an alternate charge clamp residue, Glu893, pointing towards adaptability in AR AF-2 charge clamp formation.
Sequence alignment of NR coactivator sequences shows that positively charged residues are favored N-terminal to the core hydrophobic motif while negatively charged residues are favored C-terminal to the motif (He and Wilson 2003
). Our phage-selected peptides are consistent with this trend. Arginines and lysines were observed at the N-terminal −1 position in all peptides, except for LxxLL, in which Arg was present at the −3 position. Moreover, four out of seven peptides contained negatively charged aspartate or glutamate residues C-terminal to the core motif. While previous studies have shown that complementary interactions between charged residues flanking coactivator signature motifs of coactivators and charged residues surrounding the AF-2 cleft modulated binding to the receptor (He and Wilson 2003
), we find that the flanking charged residues are typically disordered in the electron density, with only Arg−1 of FxxLF interacting with Glu897, and Lys+2 of FxxYF forming a water-mediated hydrogen bond to Asp731. Thus, if charge–charge interactions between flanking peptide residues and the AR surface occur, they are too weak to be detected crystallographically.
Finally, the AR AF-2 surface is an attractive target for pharmaceutical design. Selective peptide inhibitors that bind the AF-2 surface of liganded ERα, ERβ, and TRβ have been developed (Geistlinger and Guy 2003
), and similar α-helix–mediated protein–protein interfaces have successfully been targeted with tight binding small molecule inhibitors (Asada et al. 2003
; Vassilev et al. 2004
). Drugs that directly interfere with coactivator binding or formation of the AR N/C interaction would likely inhibit AR activity, perhaps even in androgen-resistant prostate cancers in which conventional therapies have failed. Strategies for designing AR coactivator antagonists are revealed in spite of the changes to the structure at the interface. Together the +1, +4, and +5 subsites contribute the majority of buried surface area of the peptide–LBD interaction (). Inhibitors may be designed by varying hydrophobic constituents at these hotspots. The +1 and +5 subsites of AR have a unique preference for aromatic side chains and provide the most viable starting points for designing AR-specific inhibitors. Aromatic groups, possibly with polar constituents to exploit hydrogen bonding interactions with Gln733 and Gln738 in the +1 and +5 subsites, respectively, may provide promising leads. Indeed, initial screens have yielded compounds that bind to the +1 subsite in such a manner (E. Estébanez-Perpiñá, personal communication). Poorly conserved binding and a lack of strong structural features at the +4 subsite suggest that this site may be incorporated for achieving other characteristics important for inhibitors besides fit. Synthetic strategies that link together groups that bind with moderate affinity to the +1, +5, and possibly +4 subsites may yield tight binding inhibitors of AR coactivator association.