Sequence coevolution between RNA and protein characterized by mutual information between residue triplets

Abstract

Coevolving residues in a multiple sequence alignment provide evolutionary clues of biophysical interactions in 3D structure. Despite a rich literature describing amino acid coevolution within or between proteins and nucleic acid coevolution within RNA, to date there has been no direct evidence of coevolution between protein and RNA. The ribosome, a structurally conserved macromolecular machine composed of over 50 interacting protein and RNA chains, provides a natural example of RNA/protein interactions that likely coevolved. We provide the first direct evidence of RNA/protein coevolution by characterizing the mutual information in residue triplets from a multiple sequence alignment of ribosomal protein L22 and neighboring 23S RNA. We define residue triplets as three positions in the multiple sequence alignment, where one position is from the 23S RNA and two positions are from the L22 protein. We show that residue triplets with high mutual information are more likely than residue doublets to be proximal in 3D space. Some high mutual information residue triplets cluster in a connected series across the L22 protein structure, similar to patterns seen in protein coevolution. We also describe RNA nucleotides for which switching from one nucleotide to another (or between purines and pyrimidines) results in a change in amino acid distribution for proximal amino acid positions. Multiple crystal structures for evolutionarily distinct ribosome species can provide structural evidence for these differences. For one residue triplet, a pyrimidine in one species is a purine in another, and RNA/protein hydrogen bonds are present in one species but not the other. The results provide the first direct evidence of RNA/protein coevolution by using higher order mutual information, suggesting that biophysical constraints on interacting RNA and protein chains are indeed a driving force in their evolution.

Figure 1. Likelihood of contact between triplets (orange *, red o) and pairs (blue □, green ∇) of residue positions vs. the coevolution rank.

High MI triplets are likely to be in contact for triplets of residue positions (similar to coevolution seen in protein position pairs [5]), but doublets are not. For example, 40% of the top 5 highest ranking MI triplets are in contact, while only 20% of the top 5 highest ranking MI doublets are in contact. MI between RNA and polar amino acids, more likely to lie on the surface on the protein and therefore interact with RNA, enhances the trend (o triplets, ∇ doublets). High MI triplets between polar amino acids and RNA are most likely to be in contact. In comparison, random coevolution between pairs of amino acids is expected to have a contact frequency of 8% .

Figure 2. The top ten highest MI triplets for U519 (2A, red) and C487 (2B, yellow) form a connected series across the protein structure (standard E.coli numbering).

Both U519 and C487 form base pairs with other 23S nucleotides, and thus all interactions are via backbone atoms. Many of the amino acids in the high MI clusters are at the edges of secondary structure elements. Other nucleotides did not have high MI triplets clustered in 3D space.

Figure 3. Residue distributions for the most proximal high MI triplet with U519, U519/R18/D22, a typical high MI proximal triplet.

A pyrimidine (RNA is C or U) results in a tight distribution in which R18 is an Arg (R) and D22 is an Asp (D). A purine (RNA is A or G), slightly smaller than pyrimidines, results in a more diverse distribution in which R18 is most commonly an Asn (N) or Arg (R) and D22 is more widely distributed.

Figure 4. Structural evidence explains the residue distributions for triplet U519/R18/D22.

In E.coli, the values of RNA U519/L22 R18/L22 D22 are U, Arg (R) and Asp (D), respectively. A hydrogen bond network in E.coli goes from the side chain of D22 to the side chain of R18 to the phosphate atom of U519. (Figure 4A), and explains the tight coupling seen in the distribution (Figure 3). The triplet from the archeon haloarcula marismortui represents a shift from pyrimidine to purine, with the values of U519/R18/D22 at G, Lys (K) and Arg (R), respectively. A structural alignment of the crystal structure from both species reveals that the hydrogen bonds are broken when the RNA is a purine and the residues farther apart (Figure 4B). This data suggests that the change in packing to accommodate a larger RNA side chain influences the packing between the L22 and 23S protein in such a way that this hydrogen bond network is broken.