Communication
Coupled Unfolding and Dimerization by the PAH2 Domain of the Mammalian Sin3A Corepressor

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Abstract

Coregulator recruitment by sequence-specific DNA binding transcription factors constitutes an important step in many eukaryotic transcription regulatory pathways. The Sin3 corepressor is an evolutionarily conserved protein and a key component of a large histone deacetylase-associated corepressor complex. The Sin3 corepressor contains four imperfect repeats of a domain called PAH (paired amphipathic helix) that serve as docking sites for a variety of sequence-specific DNA binding factors and coregulators. At least two closely related Sin3 proteins designated Sin3A and Sin3B have been described in higher organisms and although functional differences between these paralogs are only beginning to be appreciated, differences at the structural level are poorly understood. Here we analyze the conformational properties of the apo form of the mammalian Sin3A (mSin3A) PAH2 domain. At low micromolar concentrations, the domain is predominantly monomeric and folded in a conformation similar to those found in complexes with the Mad1 and HBP1 repressors. Unexpectedly, at higher concentrations, the domain dimerizes with concomitant population of a partially unfolded conformer. These findings are in contrast to those reported for the mSin3B PAH2 domain and may have implications for the manner in which these paralogous domains interact with their targets.

Section snippets

NMR studies of the mSin3A PAH2 domain

The construct for the mSin3A PAH2 domain (spanning residues 295–383) used for the NMR studies contains 85 non-proline residues. At least 146 backbone correlations could be readily identified in the 1H-15N heteronuclear single quantum coherence (HSQC) spectrum of a 1 mM mSin3A PAH2 sample, indicative of two conformations in slow exchange on the NMR timescale (Figure 1(a)). The HSQC spectrum recorded at 0.05 mM sample concentration is characterized by a strong reduction in intensity of a large

Backbone resonance assignments for the apo-mSin3A PAH2 domain

To characterize the polypeptide backbone conformation of the two conformationally distinct species of the mSin3A PAH2 domain, we assigned the backbone 1H, 13C, and 15N resonances using standard double and triple-resonance approaches. To increase the reliability of the assignments and to resolve ambiguities, these experiments were conducted at 0.25 mM and 1.0 mM protein concentrations where the relative populations of the two species differ significantly. We designate the predominant conformer

Analytical ultracentrifugation analysis of the mSin3A PAH2 domain

Many correlations in the 1H-15N HSQC spectra of the mSin3A PAH2 domain move progressively as a function of concentration, implying possible reversible self-association with fast dissociation kinetics (center panels, Figure 1). To test this possibility, we conducted sedimentation equilibrium experiments. A broad range of loading concentrations and wavelengths were selected for these measurements to assure significant contributions from monomeric and possibly higher order oligomeric species. Data

An integrated model for the apo-mSin3A PAH2 domain

Since the apo-mSin3A PAH2 domain exhibits a number of unusual properties, we summarize the key features and present plausible models to explain these results. The domain exists in at least two distinct conformational states including a folded monomer (the A-form) that is in equilibrium with a partially unfolded form (the B-form). The equilibrium is weighted heavily towards the A-form at low protein concentrations. Additionally, the domain undergoes reversible self-association, although the

Acknowledgements

We thank Jonathan Widom for useful discussions relating to coupled equilibria, and Richard Kang and Joanne Mai for their early contributions towards the development of this work. This work was supported by a grant from the NIH (GM 64715) to I.R. The development of the UltraScan software was supported by the NSF through a grant (DBI-9974819) to B.D. I.R. is a Scholar of the Leukemia and Lymphoma Society. Z.Z. was supported in part by a summer research scholarship from Northwestern University.

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    Y.Z. and Z.Z. contributed equally and should be considered co-first authors.

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