Figure 1.
Inter-histone H3-H3′ interface is destabilized by mutagenesis.
Models for H3-H3′ interface mutations that were tested in this study (right panels) are shown in comparison to the wild type interface (left panels). One of the binding partners is shown in surface representation, while the other is shown in cartoon representation with spheres depicting the mutated residues. H113A (A) results in the loss of a hydrogen bond with D123 across the interface, L126A (B), results in loss of hydrophobic interactions across the interface, A114Y (C) introduces bulky side-chain in the interface, and L130A (D) also results in loss of hydrophobic contacts across the interface. The structures were rendered using PyMOL (http://www.pymol.org).
Figure 2.
Thermodynamic destabilization of the histone octamer correlates with lethal phenotypes in yeast.
A, Yeast strains bearing H3-H3′ interface mutations predicted to cause thermodynamic destabilization of the nucleosome are lethal. The WZY42 histone shuffle strain transformed with the wild-type or indicated H3 mutant was plated in 10-fold serial dilution on selective synthetic complete-Trp media with (right plate) or without (left plate) 5-Fluoroorotic acid (5-FOA). B, Broad analysis of growth phenotypes and their relation to predicted nucleosome stability reveal significant difference in ΔΔG between viable and lethal mutants. Box plots are shown, which represent range between 25 and 75 percentile values. Horizontal line inside the box represents the median. Whiskers correspond to values nearest to 1.5 times the interquartile range and outliers are represented as circles. P-value is obtained from two sample, single-tailed t-test. The dashed-line represents ΔΔG of 3kcal/mol, used to distinguish between stabilizing and destabilizing mutants. C, Venn diagram showing the significant overlap that exists between lethal and destabilizing mutants found in H3 and H4 for interface and buried residues. Compilation of lethal mutant results is from HistoneHits database. The numbers inside the Venn diagram refer to number of mutations belonging to the corresponding categories. D, Venn diagram showing the significant overlap that exists between viable and stabilizing mutants found in H3 and H4 for interface and buried residues. Compilation of lethal mutant results is from HistoneHits database. The numbers inside the Venn diagram refer to number of mutations belonging to the corresponding categories.
Figure 3.
Evolutionary and calculated sequence entropies have significant correlation in H4 but no correlation in H3.
A, C, Sequence logo (http://weblogo.berkeley.edu/) of propensities of different amino acids calculated using Medusa at each of the buried/interface residues show that Medusa recapitulates 75% of positions in H3 (A, top logo) and 54.5% of positions in H4 (C, top logo). For comparison, the corresponding sequence logo from evolutionary conservation (obtained from homology-derived secondary structure of proteins (HSSP) database) is also shown for H3 (A, bottom logo) and H4 (C, bottom logo). The amino acids are colored according to their physical property (hydrophobic amino acids are colored black, negatively charged red and so on). The secondary structure corresponding to each amino acid is shown at the top: helix (H), beta strand (E), hydrogen bonded turn (T), bend (S) or no secondary structure (-). B, D, Plotting positional entropy of buried residues in H3 and H4 calculated using Medusa against evolutionary positional entropy shows no correlation for H3 (B) and significant correlation for H4 (D). Each point in the plots represents a specific buried/interface residue. The actual values of Evolutionary and Medusa entropies are represented in Supplementary Tables 3 and 4.
Table 1.
List of mutations in H3 screened in this study.
Figure 4.
Coevolution of spatially remote and proximal pairs of residues in H3 suggest a function of the buried H3 residues independent of mediating stability.
Significantly coevolving pairs of residues are shown in stick representation with dotted line between their Cβ atoms. Buried/interface residues are colored blue, while the rest of the protein is colored in grey and shown using the cartoon representation. The dotted lines between residues that are spatially proximal are colored orange, while the dotted lines between spatially distant residues are colored blue. The structure was rendered using PyMOL (http://www.pymol.org).
Table 2.
List of residues coevolving in H3.
Table 3.
Correlation between evolutionary entropy and Medusa entropy in highly conserved proteins.