Thermodynamic stability of histone H3 is a necessary but not sufficient driving force for its evolutionary conservation

Abstract

Determining the forces that conserve amino acid positions in proteins across species is a fundamental pursuit of molecular evolution. Evolutionary conservation is driven by either a protein's function or its thermodynamic stability. Highly conserved histone proteins offer a platform to evaluate these driving forces. While the conservation of histone H3 and H4 "tail" domains and surface residues are driven by functional importance, the driving force behind the conservation of buried histone residues has not been examined. Using a computational approach, we determined the thermodynamically preferred amino acids at each buried position in H3 and H4. In agreement with what is normally observed in proteins, we find a significant correlation between thermodynamic stability and evolutionary conservation in the buried residues in H4. In striking contrast, we find that thermodynamic stability of buried H3 residues does not correlate with evolutionary conservation. Given that these H3 residues are not post-translationally modified and only regulate H3-H3 and H3-H4 stabilizing interactions, our data imply an unknown function responsible for driving conservation of these buried H3 residues.

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.

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).