Histones and histone variant families in prokaryotes

Abstract

Histones are important chromatin-organizing proteins in eukaryotes and archaea. They form superhelical structures around which DNA is wrapped. Recent studies have shown that some archaea and bacteria contain alternative histones that exhibit different DNA binding properties, in addition to highly divergent sequences. However, the vast majority of these histones are identified in metagenomes and thus are difficult to study in vivo. The recent revolutionary breakthroughs in computational protein structure prediction by AlphaFold2 and RoseTTAfold allow for unprecedented insights into the potential function and structure of previously uncharacterized proteins. Here, we categorize the prokaryotic histone space into 17 distinct groups based on AlphaFold2 predictions. We identify a superfamily of histones, termed α3 histones, which are common in archaea and present in several bacteria. Importantly, we establish the existence of a large family of histones throughout archaea and in some bacteriophages that, instead of wrapping DNA, bridge DNA, thereby diverging from conventional nucleosomal histones.

Fig. 1. The conventional histone protein forms nucleosomes.

a The histone fold (PDB: 1A7W). The N-terminal α1, central α2, and C-terminal α3 helices are colored blue, orange, and green respectively. Linkers L1 and L2 connect the α1 and α3 helices to the central α2 helix. b The histone dimer binds DNA at its α1 face (PDB: 5T5K). c The eukaryotic H3-H4 tetramer (PDB: 1AOI). Only the core histone fold is visualized. d The eukaryotic octameric nucleosome (PDB: 1AOI).

Fig. 2. Alternative histones are diverse and found across prokaryotes.

a Clustered sequence space of prokaryotic histones. Clustering was performed with CLANS. The color of each line indicates the sequence similarity between the two sequences; sequences that are connected by darker lines are more similar than those connected by lighter lines. Clusters are colored based on the histone category to which they belong as determined by the AlphaFold2 predictions. For a short description of each histone category, see Supplementary Table 2. b Cladogram of archaea showing the distribution of nucleosomal (Nuc), face-to-face (FtF), coiled-coil (CC), and Methanococcales (Mc) histones across different phyla. The cladogram is based on GTDB version 207. For reference, Methanobacteriota A contains the Methanopyri and Methanococci classes; Methanobacteriota B contains the Thermococci and the Methanofastidiosa classes.

Fig. 3. The face-to-face (FtF) histones form a unique tetramer structure.

a The homotetramer of FtF histone D4GZE0 from Haloferax volcanii as predicted by AlphaFold2. Each residue is colored by its predicted local distance difference test (pLDDT) value. AlphaFold2 is confident in the local structure if the pLDDT is  >70. b The conserved RxTxxxxD motif, DNA binding residues K11, and tetramerization residue N45 of FtF histone D4GZE0. K11, N45, R47, T49, and D54 relate to K16, N52, R54, T56, and D61 in the FtF HMM logo (Supplementary Fig. 9). c The `back' of the dimer-dimer interface of FtF histone D4GZE0. R41 relates to R48 in the FtF HMM logo (Supplementary Fig. 9). d Crystal structure of FtF histone HTkC from Thermococcus kodakarensis (PDB: 9F2C). e Our proposed model for how FtF histones bind and wrap DNA.

Fig. 4. ZZ-type zinc finger histones can possibly bind two zinc ions.

a The ZZ-histone D0LYE7 from Haliangium ochraceum SMP-2 as predicted by AlphaFold2. ZZ-histones contain a ZZ-type zinc finger domain at the N-terminus and an α3 histone fold at the C-terminus. Each residue is colored by its pLDDT value. b The ZZ domain of D0LYE7. This domain contains two zinc binding motifs, one C4 motif (C20, C23, C51, C53) and a C2H2 motif (C36, C41, H59, H65). These residues correspond to C10, C13, C41, and C43 for the C4 motif and C26, C31, H49, and H55 for the C2H2 motif in the HMM profile (Supplementary Fig. 18). Each residue is colored by its pLDDT value. c The ZZ domain of HERC2 (gray, PDB: 6WW4) aligned to D0LYE7.

Fig. 5. α3 histones from bacteriophages or with eukaryotic-like domains.

a The homodimer of Rab GTPase histone A0A0F8XJF6 as predicted by AlphaFold2. Rab GTPase histones contain a small Rab GTPase domain on the N-terminus and an FtF-like histone fold at the C-terminus. Each residue is colored by its pLDDT value. b The homotetramer of phage histone A0A2E7QIQ9 as predicted by AlphaFold2. Phage histones contain an α3 histone fold at the N-terminus and an α-helix which functions as the tetramerization domain at the C-terminus.

Fig. 6. DNA-bridging coiled-coil (CC) histones are widely found in archaea.

a Phylogenetic tree of CC histones. Clades are colored by phylum as they are assigned in the GTDB database (v207). The tree was generated with RAxML-NG. 1000 bootstraps were performed and used to calculate the transfer bootstrap expectation values (TBE). b The homotetramer of CC histone E3GZL0 from Methanothermus fervidus as predicted by AlphaFold2. CC histones contain a long α-helix on the C-terminus. Each residue is colored by its pLDDT value. c The homotetramer of RdgC histone D4GVY1 from Haloferax volcanii as predicted by AlphaFold2. RdgC histones form very similar tetramer structures to coiled-coil histones despite low sequence identity. Each residue is colored by its pLDDT value.

Fig. 7. IHF histones are functionally related to IHF.

a The homodimer of IHF histone A0A358AGI2 from Candidatus Omnitrophica as predicted by AlphaFold2. Each residue is colored by its pLDDT value. b Gene cluster comparison of bacterial metagenomes that contain the IHF histone. The organism and its genome ID are noted on the left. The IHF histone, IHF-like, and topoisomerase VI-like genes are colored green, orange, and blue respectively. c The monomer of IHF-like A0A358AGI6 from Candidatus Omnitrophica as predicted by AlphaFold2. Each residue is colored by its pLDDT value. Residue R45 is highlighted in green. d The RxTxxxxD motif and the “sprocket” R32 of IHF histone A0A358AGI2. R32, R65, T67, and D72 relate to R24, R57, T59, and D64 in the IHF histone HMM logo (Supplementary Fig. 34).