Biomedical subjects

David L Bain

Publications and source records attributed to David L Bain.

7 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↗

PubMed2005

Self-association energetics of an intact, full-length nuclear receptor: the B-isoform of human progesterone receptor dimerizes in the micromolar range.

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 degrees 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.

Dimerization↗

PubMed2003

Trimeric structure for an essential protein in L1 retrotransposition.

Two proteins are encoded by the mammalian retrotransposon long interspersed nuclear element 1 (LINE-1 or L1); both are essential for retrotransposition. The function of the protein encoded by the 5'-most ORF, ORF1p, is incompletely understood, although the ORF1p from mouse L1 is known to bind single-stranded nucleic acids and function as a nucleic acid chaperone. ORF1p self-associates by means of a long coiled-coil domain in the N-terminal region of the protein, and the basic, C-terminal region (C-1/3 domain) contains the nucleic acid binding activity. The full-length and C-1/3 domains of ORF1p were purified to near homogeneity then analyzed by gel filtration chromatography and analytical ultracentrifugation. Both proteins were structurally homogeneous and asymmetric in solution, with the full-length version forming a stable trimer and the C-1/3 domain remaining a monomer. Examination of the full-length protein by atomic force microscopy revealed an asymmetric dumbbell shape, congruent with the chromatography and ultracentrifugation results. These structural features are compatible with the nucleic acid binding and chaperone activities of L1 ORF1p and offer further insight into the functions of this unique protein during LINE-1 retrotransposition.

Animals↗

PubMed2003

Functional properties of the N-terminal region of progesterone receptors and their mechanistic relationship to structure.

Progesterone receptors (PR) are present in two isoforms, PR-A and PR-B. The B-upstream segment (BUS) of PR-B is a 164 amino acid N-terminal extension that is missing in PR-A and is responsible for the functional differences reported between the two isoforms. BUS contains an activation function (AF3) which is defined by a core domain between residues 54-154 whose activity is dependent upon a single Trp residue and two LXXLL motifs. We have also identified sites both within and outside of BUS that repress the strong synergism between AF3 and AF1 in the N-terminal region and AF2 in the hormone binding domain. One of these repressor sites is a consensus binding motif for the small ubiquitin-like modifier protein, SUMO-1 (387IKEE). The DNA binding domain (DBD) structure is also important for function. When BUS is linked to the glucocorticoid receptor DBD, AF3 activity is substantially attenuated, suggesting that binding to a DNA response element results in allosteric communication between the DBD and N-terminal functional regions. Lastly, biochemical and biophysical analyses of highly purified PR-B and PR-A N-terminal regions reveal that they are unstructured unless the DBD is present. Thus, the DBD stabilizes N-terminal structure. We propose a model in which the DBD through DNA binding, and BUS through protein-protein interactions, stabilize active receptor conformers within an ensemble distribution of active and inactive conformational states. This would explain why PR-B are stronger transactivators than PR-A.

Amino Acid Sequence↗

PubMed2002

Structural and conformational analysis of the oxidase to dehydrogenase conversion of xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) is a 300-kDa homodimer that can exist as an NAD+-dependent dehydrogenase (XD) or as an O2-dependent oxidase (XO) depending on the oxidation state of its cysteine thiols. Both XD and XO undergo limited cleavage by chymotrypsin and trypsin. Trypsin selectively cleaved both enzyme forms at Lys184, while chymotrypsin cleaved XD primarily at Met181 but cleaved XO at Met181 and at Phe560. Chymotrypsin, but not trypsin, cleavage also prevented the reductive conversion of XO to XD; thus the region surrounding Phe560 appears to be important in the interconversion of the two forms. Size exclusion chromatography showed that disulfide bond formation reduced the hydrodynamic volume of the enzyme, and two-dimensional gel electrophoresis of chymotrypsin-digested XO showed significant, disulfide bond-mediated, conformational heterogeneity in the N-terminal third of the enzyme but no evidence of disulfide bonds between the N-terminal and C-terminal regions or between XOR subunits. These results indicate that intrasubunit disulfide bond formation leads to a global conformational change in XOR that results in the exposure of the region surrounding Phe560. Conformational changes within this region in turn appear to play a critical role in the interconversion between the XD and XO forms of the enzyme.

Animals↗