Self-association energetics of an intact, full-length nuclear receptor: the B-isoform of human progesterone receptor dimerizes in the micromolar range.
Self-association energetics of an intact, full-length nuclear receptor: the B-isoform of human progesterone receptor dimerizes in the micromolar range.
复制标题
完整、全长核受体的自缔合能量学：人孕酮受体的 B 异构体在微摩尔范围内二聚化。
DOI：
10.1021/bi050609i
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Bain,DavidL
中科院分区：
文献类型：
--
作者：
Heneghan,AaronF;Berton,Nancy;Miura,MichaelT;Bain,DavidL
We are focused on understanding the mechanisms underlying eukaryotic gene regulation, using the human progesterone receptor (PR) and its interactions with its DNA response elements as a model system. An understanding of PR function is complicated by the presence of two transcriptionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The two isoforms are identical except the B-receptor contains an additional 164 residues at its N-terminus. As a first step toward understanding the principles by which the two isoforms assemble at complex promoters, we examined the energetics of PR-B self-association using sedimentation velocity and sedimentation equilibrium methods. Full-length human PR-B was purified to 95% homogeneity from baculovirus-infected insect cells. Using a fluorescence hormone binding assay, we determined the purified protein to be highly active in its ability to bind ligand. Sedimentation velocity studies of hormone-bound PR-B at pH 8.0, 4 °C, and 50 mM NaCl demonstrate that it undergoes a concentration-dependent change in its sedimentation coefficient, existing as a 4.0S species at submicromolar concentrations but forming a 5.7S species at higher concentrations. These results strongly suggest that PR-B undergoes self-association in the micromolar range. This hypothesis was examined rigorously using sedimentation equilibrium. Global analysis of the sedimentation equilibrium data demonstrated that PR-B self-association was well described by a monomer−dimer model with a dimerization free energy of −7.2 ± 0.7 kcal/mol. The role of NaCl in regulating PR-B dimerization was examined by carrying out sedimentation velocity and equilibrium studies under high salt conditions. At 300 mM NaCl, PR-B is exclusively monomeric in the micromolar range, thus revealing a significant ionic contribution to the assembly energetics. Further, the monomer sediments as a structurally homogeneous, but highly asymmetric, 4.0S species. Limited proteolysis of PR-B demonstrated that the hydrodynamic asymmetry is due in part to an extended, nonglobular conformation localized to the N-terminal region of PR-B. In contrast, the DNA binding domain (DBD) and hormone binding domain (HBD) exist as independent structural units, and the activation function N-terminal to the DBD (AF-1) shows moderate structure. These results represent the first rigorous analysis of the self-assembly energetics of an intact nuclear receptor and suggest that PR function is more complex than envisioned by traditional models.
