title: Hydrodynamic analysis of the human progesterone receptor A-isoform reveals that self-association occurs in the micromolar range.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Connaghan-Jones
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Heneghan
authors.forename: Michael T
authors.initials: MT
authors.lastname: Miura
authors.forename: David L
authors.initials: DL
authors.lastname: Bain
mesh_terms: D019281:Dimerization; D006801:Humans; D020033:Protein Isoforms / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011980:Receptors, Progesterone / Q000737:chemistry* / Q000235:genetics; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D020033:Protein Isoforms; D011980:Receptors, Progesterone