The N-terminal Regions of Estrogen Receptor (cid:1) and (cid:2) Are Unstructured in Vitro and Show Different TBP Binding Properties*

The N-terminal regions of the estrogen receptor (cid:1) (ER (cid:1) -N) and (cid:2) (ER (cid:2) -N) were expressed and purified to homogeneity. Using NMR and circular dichroism spectroscopy, we conclude that both ER (cid:1) -N and ER (cid:2) -N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ER (cid:1) -N in vitro and to potentiate ER-activated transcription. We used surface plasmon resonance and circular dichroism spectroscopy to confirm and further characterize the ER-N-TBP interaction. Our results show that the intrinsically unstructured ER (cid:1) -N interacts with TBP, and suggest that structural changes are induced in ER (cid:1) -N upon TBP interaction. Conformational changes upon target factor interaction have not previously been demonstrated for any N-terminal region of nuclear receptors. In addition, no binding of ER (cid:2) -N to TBP was detected. This difference in TBP binding could imply differential recruitment of target fitted to a linear model assuming first order kinetics and with nonco- operative one-to-one binding of ER (cid:1) -N to TBP. The kinetics of this interaction is described by the rate equation d R /d t (cid:5) k a (cid:6) C (cid:6) R max (cid:4) ( k a (cid:6) C (cid:7) k d ) (cid:6) R . Here, k a and k d are association and dissociation rates, respectively, and C is the concentration of injected interactant. The response, R , is assumed to be proportional to the amount of bound analyte and R max is the response when maximal binding capacity is reached. Thereafter, data were also fitted to a conformational change model (see description by Wa¨rnmark et al. in Ref. 31).