Role of Structural and Dynamical Plasticity in Sin3: The Free PAH2 Domain is a Folded Module in mSin3B Hugo van Ingen, Maria A. H. Baltussen, Jan Aelen and Geerten W. Vuister * Department of Physical Chemistry/Biophysical Chemistry, Institute for Molecules and Materials Radboud University Nijmegen Toernooiveld 1, 6525 ED Nijmegen, the Netherlands The co-repressor Sin3 is the essential scaffold protein of the Sin3/HDAC co- repressor complex, which is recruited to the DNA by a diverse group of transcriptional repressors, targeting genes involved in the regulation of the cell cycle, proliferation and differentiation. Sin3 contains four repeats commonly denoted as paired amphipathic helix (PAH1-4) domains that provide the principal interaction surface for various repressors. Here, we present the first structure of the free state of the PAH2 domain and discuss its implications for interaction with the repressors. The unbound conformation is very similar to the conformation observed when bound to either the Mad1 or HBP1 repressor, suggesting that the PAH2 domain serves as a template that guides proper folding of the unstructured repressor. The free PAH2 domain shows micro- to millisecond confor- mational exchange between the folded, major state and a partially unfolded, minor state. Upon complex formation, we observe a significant decrease in fast time-scale flexibility of local regions of the protein, correlated with the formation of intermolecular contacts, and an overall decrease in the slow time-scale conformational exchange. On the basis of our data and using a multiple sequence alignment of all PAH domains, we suggest that the PAH1, PAH2 and PAH3 domains form pre-folded binding modules in full-length Sin3 like beads-on-a-string, and act as folding templates for the interaction domains of their targets. q 2006 Elsevier Ltd. All rights reserved. Keywords: protein–protein interactions; PAH domain; transcription; Sin3; NMR *Corresponding author Introduction The regulation of gene expression in eukaryotes requires the formation of specific protein–DNA and protein–protein complexes. 1 Transcriptional activa- tors and repressors recognize specific DNA sequences upstream of the promoter of target genes, resulting in either up-regulation or down- regulation of transcription. 2,3 This is achieved indirectly via recruitment of co-regulators and associated proteins that together are capable of shaping the chromatin structure into an active or repressive state. 4–7 The association between the activator/repressor and the co-regulator is a crucial step, as it provides the link between target gene and chromatin modification. A diverse group of repressors recruit a co- regulator complex containing the highly conserved co-repressor Sin3, 8 and the class I histone deacety- lating proteins (HDAC1/2). The HDAC proteins deacetylate specific lysine residues in the tails of the histone proteins, resulting in a compact, repressive chromatin state. 9–13 The core Sin3/HDAC complex further consists of Sin3 associated proteins (SAP18 and SAP30), that are thought to stabilize the complex, 11,14 and histone-targeting proteins (RbAp48 and RbAp46), that stabilize contact with the nucleosome. 9,11 Additionally, the core Sin3/ HDAC co-repressor complex exists of SDS3, 15 SAP130 and SAP180 16 and can be extended with 0022-2836/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. Present address: M.A.H. Baltussen, RIKILT, Institute of Food Safety, Wageningen, The Netherlands. Abbreviations used: CPMG, Carr–Purcell–Meiboom– Gill; HMG, high mobility group; NOE, nuclear Over- hauser effect; O-GlcNAc, O-linked N-acetylglucosamine; RMSD, root-mean-square deviation; PDB, Protein Data Bank; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; HSQC, heteronuclear single quantum coherence. E-mail address of the corresponding author: g.vuister@science.ru.nl doi:10.1016/j.jmb.2006.01.100 J. Mol. Biol. (2006) 358, 485–497