A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, a polyubiquitin is found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition.
- Volker Dötsch, Goethe University, Germany
© 2015, Liu et al.
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Genome and epigenome integrity in eukaryotes depends on the proper coupling of histone deposition with DNA synthesis. This process relies on the evolutionary conserved histone chaperone CAF-1 for which the links between structure and functions are still a puzzle. While studies of the Saccharomyces cerevisiae CAF-1 complex enabled to propose a model for the histone deposition mechanism, we still lack a framework to demonstrate its generality and in particular, how its interaction with the polymerase accessory factor PCNA is operating. Here, we reconstituted a complete SpCAF-1 from fission yeast. We characterized its dynamic structure using NMR, SAXS and molecular modeling together with in vitro and in vivo functional studies on rationally designed interaction mutants. Importantly, we identify the unfolded nature of the acidic domain which folds up when binding to histones. We also show how the long KER helix mediates DNA binding and stimulates SpCAF-1 association with PCNA. Our study highlights how the organization of CAF-1 comprising both disordered regions and folded modules enables the dynamics of multiple interactions to promote synthesis-coupled histone deposition essential for its DNA replication, heterochromatin maintenance, and genome stability functions.
Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized ‘codes’ that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the ‘histone code’ concept and interrogate it at the nucleosome level.