Journal of Molecular Biology
CommunicationCoupled Unfolding and Dimerization by the PAH2 Domain of the Mammalian Sin3A Corepressor
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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Cited by (12)
Aa-Hub domains and intrinsically disordered proteins: A decisive combo
2021, Journal of Biological ChemistryCitation Excerpt :In a study addressing binding of Sin3 isoforms, Sin3a-PAH2 and Sin3b-PAH2 bound Pf1 with comparable affinities but apparently different thermodynamic profiles. This likely reflects that apo-Sin3a-PAH2 samples both folded and partially folded conformations and forms a monomer–dimer equilibrium and that apo-Sin3b-PAH2 is monomeric and mostly folded (see below) (43, 46, 118, 119). Accordingly, Sin3a-PAH2 and HBP1 undergo mutual coupled conformational transitions upon association (44).
Sin3: Insight into its transcription regulatory functions
2013, European Journal of Cell BiologyCitation Excerpt :Unlike the other PAH domains, PAH4 most likely does not fold as a four-helix bundle, but instead adapts a distinct fold (van Ingen et al., 2006). Structural studies have revealed that the PAH2 domain of mammalian Sin3A exhibits conformational heterogeneity that enables Sin3A to regulate its interaction with diverse protein targets (Brubaker et al., 2000; Swanson et al., 2004; Zhang et al., 2006). Although, there is a high degree of similarity between the PAH1 and PAH2 domains, PAH1 recognizes different sequence motifs as compared to PAH2 thereby enabling a high degree of target specificity (Le Guezennec et al., 2006; Sahu et al., 2008).
Solution structure of the mSin3A PAH2-Pf1 SID1 complex: A Mad1/Mxd1-like interaction disrupted by MRG15 in the Rpd3S/Sin3S complex
2011, Journal of Molecular BiologyCitation Excerpt :Finally, Pf1 SID1 is not the only interactor that exhibits conformational plasticity; a subset of the mSin3A PAH2 residues at the interface and beyond, particularly in the α1 and α4 helices, also exhibits side-chain packing diversity in the various complexes.24 These are the same regions that also show conformational diversity in apo-mSin3A PAH2.41 The coding sequences of mammalian Sin3A PAH2 (residues 295–385) and human Pf1200–241 (residues 200–241) were amplified by PCR and inserted into the pMCSG21 and pMCSG7 expression vectors, respectively.
Sin3: Master scaffold and transcriptional corepressor
2009, Biochimica et Biophysica Acta - Gene Regulatory MechanismsConserved Themes in Target Recognition by the PAH1 and PAH2 Domains of the Sin3 Transcriptional Corepressor
2008, Journal of Molecular Biology
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Y.Z. and Z.Z. contributed equally and should be considered co-first authors.