Similarities between estradiol binding to human ERα and amphioxus SRIn Fig. 3A and B, we show the interaction of E2 with key residues that stabilize E2 in human ERα [24], [25] and [26] and amphioxus SR, respectively. Fig. 3A shows the hydrogen bonds between E2 and Glu-353 and Arg-394 on human ERα. The phenolic hydroxyl on E2 is 2.8 ? from Oε2 on Glu-353 and 2.9 ? from Nη2 on Arg-394. Nε on Arg-394 contacts the backbone oxygen on Phe-404. Cε2 on Phe-404 also has a van der Waals contact with C10 on estradiol. Cδ1 on Leu-525 and Cε on Met-421 have van der Waals contacts with the 17β-hydroxyl and C16, respectively, on E2 (Fig. 3A).Fig. 3. Similarities in the binding of estradiol to human ERα and amphioxus SR. (A) Interaction between estradiol and human ERα. (B) Interaction between estradiol and the 3D model of amphioxus SR.Figure optionsDownload full-size imageDownload as PowerPoint slideFig. 3B shows that in amphioxus SR the C3-hydroxyl on E2 is 2.7 ? from Oε1 on Glu-346 and 3.5 ? from Nη1 on Arg-387. Nε on Arg-387 is 4 ? from the backbone oxygen on Phe-398. Cε2 on Phe-389 has a van der Waals contact with C10 on E2. Cβ on Leu-507 and Sδ on Met-414 have van der Waals contacts with 17β-hydroxyl and C16, respectively, on E2 (Fig. 3B). The backbone oxygen of Gly-503 contacts the 17β-hydroxyl of E2.Differences between estradiol binding to human ERα and amphioxus SRThere are, however, several differences between human ERα and amphioxus SR in their interaction with E2. Fig. 4 shows the residues on human ERα and amphioxus SR that we find differ in binding E2.Fig. 4. Differences the binding of estradiol to human ERα and amphioxus SR. (A) Interaction between estradiol and human ERα. (B) Interaction between estradiol and the 3D model of amphioxus SR.Figure optionsDownload full-size imageDownload as PowerPoint slideIn human ERα, Nδ1 on His-524 is 2.8 ? from the 17β-hydroxyl on the D ring of E2. In addition, Cε1 on His-524 has a van der Waals contact with the 17β-hydroxyl on E2, which is 3.4 ? from Cε1 (not shown). His-524 also is stabilized by an interaction with the backbone oxygen on Glu-419, which is 3.3 ? from Nε2 on His-524 [25] (Fig. 4A). E2 has stabilizing contacts with Sδ of Met-343 and Cδ1 on Ile-424 (Fig. 4A).In amphioxus SR, due to a rotation of His-506, it has a different interaction with the 17β-hydroxyl on E2. Nδ1 on His-506 is 3.2 ? from the 17β-hydroxyl on E2 (Fig. 3B). Cε1 on His-506 does not have a van der Waals contact with the 17β-hydroxyl on E2. However, Cδ2 on His-506 has van der Waal contacts with the 17β-hydroxyl, C17 and C16 on E2, which are 3.6 ?, 3.9 ? and 3.5 ? distant, respectively, from Cδ2 (not shown).In amphioxus SR, Met-336, which corresponds to Met-343 on human ERα, does not have a stabilizing contact with E2. Instead, Sδ on Met-336 is 7.2 ? from the 17β-hydroxyl on E2. The backbone nitrogen of Ser-412 is 3.4 ? from Nε2 on His-506. Ile-417 does not interact with E2 (Fig. 4B).DiscussionTwo recent studies with amphioxus ER and SR have provided important insights into the ancestry of steroid
BI 2536 signaling in chordates [13] and [14]. The absence of E2 activation of amphioxus ER [13] and [14] and the activation by E2 of amphioxus SR [13] were surprising. In a cell culture assay, amphioxus SR is not as responsive to E2 as is human ERα; the SR requires about 100 nM E2 to activate gene transcription to 50% of the maximum response [13]. This is substantially higher than 0.2 nM E2 for the Kd of E2 for human ERα [15].Unexpectedly, amphioxus SR is not activated by 3-ketosteroids [13], although the SR sequence clearly clusters in a phylogenetic analysis with 3-ketosteroid receptors [13] and [14]. These intriguing findings indicate that there are unique aspects of amphioxus SR regarding steroid-binding [13]. Our 3D model of amphioxus SR begins to elucidate some of its surprising properties.Comparisons of the 3D structures of human ERα and amphioxus SR reveal that their overall 3D structures are conserved. Indeed, amphioxus SR contains key residues that are important for stabilizing binding of E2 to human ERα (Fig. 3B) [13]. In particular, Glu-346 and Arg-387 stabilize the C3-hydroxyl on E2. Moreover, His-506 has a stabilizing contact with the 17β-hydroxyl on E2. This interaction between histidine and E2 is unique to vertebrate ERs and is not found in other adrenal and sex steroid receptors [27], [28], [29], [30] and [31] including the lamprey PR and CR [9]. Also stabilizing E2 in amphioxus SR are contacts between the 17β-hydroxyl on E2 and Gly-503 and Leu-507 (Fig. 3B).There are differences in the binding of the D ring to the SR, which may contribute to the lower transcriptional activation of amphioxus SR by E2. Compared to His-524 in human ERα, His-506 has rotated and does not have the same stabilizing interactions with E2 as found in human ERα. Also in amphioxus SR, Met-336 and Ile-417 do not contact E2 (Fig. 4B).Evolutionary implicationsAmphioxus SR is intriguing because, although it binds and is activated by E2 [13], the amino
acid sequence of SR clearly places it closest to the GR and other 3-ketosteroid receptors [13] and [14]. Moreover, the SR does not bind 3-ketosteroids. This may be due to the presence of Glu-346 instead of a Gln, which acts as a hydrogen bond donor to the C3-ketone in the PR [31], GR [27], AR [30] and MR [28] and [29]. Indeed, Ekena et al. [32] showed that in human ERα, conversion of Glu-353 to Gln increases the affinity of mutant human ERα for testosterone by 140-fold, providing a parsimonious mechanism for the evolution of receptors that respond to 3-ketosteroids from an ER.E2 and the enzymes that can synthesize E2 are found in amphioxus [33] and [34]. However, physiological regulation of amphioxus SR activity by 100 nM E2 is unlikely because the E2 concentration in amphioxus is low [34]. It may be that the SR is activated by an estradiol derivative [19] and [20]or other steroids, such as Δ5-androstene-3β,17β-diol [Δ5-Adiol] and 5-androstane-3β,17β-diol [17] and [21]. Both Δ5-Adiol and 5-androstane-3β,17β-diol have Kds in the nM range for human ERα [15], and both steroids contain a C3 alcohol and a 17β-hydroxyl that can form a hydrogen bond with Glu-346 and His-506, respectively, in amphioxus SR.Δ5-Adiol, which is formed from DHEA by reduction of the C17-ketone to an alcohol can be metabolized to testosterone by 3β-hydroxysteroid dehydrogenase. Testosterone would be an inactive metabolite. However, mutation of Glu-346 to Gln in the SR may yield a mutant receptor that binds testosterone [32], providing a mechanism for the evolution of receptors for 3-ketosteroids. This hypothesis can be tested by investigating the steroid specificity of the Gln-346 mutant.
