Molecular basis of histone tail recognition by human TIP5 PHD finger and bromodomain of the chromatin remodeling complex NoRC.
Bottom Line: TIP5 domains that recognize posttranslational modifications on histones are essential for recruitment of NoRC to chromatin, but how these reader modules recognize site-specific histone tails has remained elusive.PHD finger functions as an independent structural module in recognizing unmodified H3 histone tails, and the bromodomain prefers H3 and H4 acetylation marks followed by a key basic residue, KacXXR.Further low-resolution analyses of PHD-bromodomain modules provide molecular insights into their trans histone tail recognition, required for nucleosome recruitment and transcriptional repression of the NoRC complex.
Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK; Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford OX3 7FZ, UK.Show MeSH
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Mentions: In order to examine the plasticity of this junction between reader domains in solution, we further analyzed tandem TIP5 and BAZ2B proteins (PHD-BRD) using SAXS. The scattering data were collected from free tandem domains and, for BAZ2B, also in complex with the histone H3 peptide with the K14 acetylation mark H3(1–18)K14ac (Table S1). The experimental molecular mass (MM) of both proteins (28 ± 5 kDa for TIP5 and 33 ± 5 kDa for BAZ2B) points to monomeric state of the two PHD-BRD constructs in solution. The Kratky plots (Figure S6B) for the tandems in the free state do not display a bell shape, expected for compact particles, but instead indicate an extended shape and potential flexibility. Comparing the two curves in Figure S6B, TIP5 tandem shows somewhat higher compactness, whereas BAZ2B follows a pattern closer to that of flexible proteins. These qualitative conclusions are corroborated by the overall parameters of the two constructs, radius of gyration (Rg) and maximum diameter (Dmax) (Rg = 31 ± 1 Å, Dmax = 100 ± 10 Å for TIP5; Rg = 42 ± 1 Å, Dmax = 145 ± 10 Å for BAZ2B). These parameters are indicative of extended structures, whereby TIP5 is more compact than BAZ2B. The distance distribution functions p(r) displayed skewed shapes characteristic for elongated particles (Figure S6C). To characterize the potential protein mobility, an ensemble optimization method (EOM; Bernadó et al., 2007) was used, which selects an ensemble of conformers fitting the experimental data from a large pool of generated models with random linker conformations. The Rg and Dmax distribution analyses of TIP5 from the EOM-selected ensemble (Figures 5C–5E) indicated that the ensemble is more compact than the random pool, suggesting a limited flexibility of the linker between reader domains. In contrast, the EOM results on the collected SAXS data of BAZ2B (Figures 5F–5H), showed broad Rg and Dmax distributions, which are more extended in comparison with the pool, thereby suggesting high flexibility on the linker. The combined data strongly proposed an extended and flexible linker conformation for BAZ2B and a rather more rigid and compact arrangement for TIP5. The limited flexibility of TIP5 allowed us to reconstruct its low-resolution shape ab initio and also to build a hybrid model of the construct in which the linker is represented by a chain of dummy residues. The models presented in Figures 5A and 5B fit well the experimental data and display similar overall shapes revealing the probable conformation that TIP5 can adopt in solution.
Affiliation: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK; Structural Genomics Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ, UK; Target Discovery Institute, Nuffield Department of Clinical Medicine, University of Oxford, NDM Research Building, Roosevelt Drive, Oxford OX3 7FZ, UK.