Biomedical subjects

Keith D Connaghan-Jones

Publications and source records attributed to Keith D Connaghan-Jones.

3 recordsLinked to original sources

PubMed2007

Nuclear receptor structure: implications for function.

Small lipophilic molecules such as steroidal hormones, retinoids, and free fatty acids control many of the reproductive, developmental, and metabolic processes in eukaryotes. The mediators of these effects are nuclear receptor proteins, ligand-activated transcription factors capable of regulating the expression of complex gene networks. This review addresses the structure and structural properties of nuclear receptors, focusing on the well-studied ligand-binding and DNA-binding domains as well as our still-emerging understanding of the largely unstructured N-terminal regions. To emphasize the allosteric interdependence among these subunits, a more detailed inspection of the structural properties of the human progesterone receptor is presented. Finally, this work is placed in the context of developing a quantitative and mechanistic understanding of nuclear receptor function.

Animals↗

PubMed2006

Hydrodynamic analysis of the human progesterone receptor A-isoform reveals that self-association occurs in the micromolar range.

Human progesterone receptors exist as two functionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 amino acids at its N-terminus. We have previously characterized the hydrodynamics and solution assembly energetics of PR-B [Heneghan, A. F., et al. (2005) Biochemistry 44, 9528-9537], and here we present an analysis of PR-A. At micromolar concentrations of the receptor, sedimentation velocity studies demonstrate that PR-A undergoes a concentration-dependent change in its sedimentation coefficient distribution, indicative of a self-associating system. Global analysis of data sets collected at multiple PR-A concentrations supports the presence of a hydrodynamically homogeneous 3.50 S monomer species in equilibrium with a 7.15 S dimer species. Sedimentation equilibrium analysis demonstrates that self-association can be rigorously described by a monomer-dimer assembly reaction and a dimerization free energy of -7.6 +/- 0.6 kcal/mol. Both the PR-A monomer and dimer are structurally asymmetric, although the extent of asymmetry is significantly decreased for the dimer, indicative of quaternary-induced hydrodynamic compaction. Limited proteolysis studies suggest that PR-A asymmetry arises from an ensemble of partially folded conformations within the N-terminal half of the molecule. Finally, comparison to our previous work on PR-B self-association energetics demonstrates that it dimerizes, under identical solution conditions, with an affinity at least 8-fold weaker than that of PR-A. Thus, residues unique to the B-isoform destabilize receptor assembly energetics. Importantly, the physical and chemical driving forces underlying isoform-specific dimerization suggest that B-unique amino acids modulate the energetics through an allosteric mechanism.

Dimerization↗

PubMed2006

Cooperative DNA binding by the B-isoform of human progesterone receptor: thermodynamic analysis reveals strongly favorable and unfavorable contributions to assembly.

Progesterone receptors (PR) play critical roles in eukaryotic gene regulation, yet the mechanisms by which they assemble at multisite promoters are poorly understood. Here we present a thermodynamic analysis of the interactions of the PR B-isoform (PR-B) with promoters containing either one or two progesterone response elements (PREs). Utilizing quantitative footprinting, we have resolved the microscopic energetics of PR-B binding, including cooperativity terms. The results of this analysis challenge a number of assumptions found in traditional models of receptor function. First, PR-B interactions at a single PRE can be equally well described by mechanisms invoking either the receptor monomer or the dimer as the active DNA binding species. If, as is commonly accepted, PR-B interacts with response elements only as a preformed dimer, then its intrinsic binding affinity is not the typically observed nanomolar but is rather picomolar. This high affinity binding is opposed, however, by a large energetic penalty. The penalty presumably pays for costly structural rearrangements of the receptor dimer and/or response element that are needed to form the protein-DNA complex. If PR-B assembles at a single response element via successive monomer binding reactions, then this penalty minimizes cooperative interactions between adjacent monomers. When binding to two response elements, the receptor exhibits strong intersite cooperativity. Although this phenomenon has been observed before, the present work demonstrates that the energetics reach levels seen in highly cooperative systems such as lambda cI repressor. This first quantitative dissection of cooperative receptor-promoter interactions suggests that PR-B function is more complex than traditionally envisioned.

Allosteric Site↗