The N-terminal regions of estrogen receptor alpha and beta are unstructured in vitro and show different TBP binding properties.
The N-terminal regions of the estrogen receptor alpha (ER alpha-N) and beta (ER beta-N) were expressed and purified to homogeneity. Using NMR and circular dichroism spectroscopy, we conclude that both ER alpha-N and ER beta-N are unstructured in solution. The TATA box-binding protein (TBP) has been shown previously to interact with ER alpha-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 alpha-N interacts with TBP, and suggest that structural changes are induced in ER alpha-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 beta-N to TBP was detected. This difference in TBP binding could imply differential recruitment of target proteins by ER alpha-N and ER beta-N. The affinity of the ER alpha-N-TBP interaction was determined to be in the micromolar range (K(D) = 10(-6) to 10(-5) m). Our results support models of TBP as a target protein for the N-terminal activation domain of ER alpha. Further, our results suggest that target proteins can induce and/or stabilize ordered structure in N-terminal regions of nuclear receptors upon interaction.
Wärnmark
A
A
Department of Biosciences, Novum, Karolinska Institutet, Huddinge SE-141 57, Sweden. anette.warnmark@cbt.ki.se
Wikström
A
A
Wright
A P
AP
Gustafsson
J A
JA
Härd
T
T
D001483:Base Sequence; D002461:Cell Line, Transformed; D002942:Circular Dichroism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D066298:In Vitro Techniques; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011960:Receptors, Estrogen / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D011960:Receptors, Estrogen; D035181:TATA-Box Binding Protein; D014157:Transcription Factors