Complementary advantageous substitutions in the evolution of an antiviral RNase of higher primates

Abstract

An improved understanding of the evolution of gene function at the molecular level may provide significant insights into the origin of biological novelty and adaptation. With the approach of ancestral protein reconstruction, we here address the question of how a dramatically enhanced ribonucleolytic activity and the related antiviral activity evolved in a recently duplicated ribonuclease (eosinophil-derived neurotoxin) gene of higher primates. We show that the mother gene of the duplicated genes had already possessed a weak antiviral activity before duplication. After duplication, substitutions at two interacting sites (Arg-64-->Ser and Thr-132-->Arg) resulted in a 13-fold enhancement of the ribonucleolytic activity of eosinophil-derived neurotoxin. These substitutions are also necessary for the potent antiviral activity, with contributions from additional amino acid changes at interacting sites. Our observation that a change in eosinophil-derived neurotoxin function occurs only when both interacting sites are altered indicates the importance of complementary substitutions in protein evolution. Thus, neutral substitutions are not simply "noises" in protein evolution, as many have thought. They may play constructive roles by setting the intramolecular microenvironment for further complementary advantageous substitutions, which can lead to improved or altered function. Overall, our study illustrates the power of the "paleomolecular biochemistry" approach in delineating the complex interplays of amino acid substitutions in evolution and in identifying the molecular basis of biological innovation.

Figure 1

Gene duplication and functional changes in the evolution of EDN and ECP genes of higher primates. The RNase activities shown are from the present study, except that of ECP, which was transformed from the originally reported value to reflect the activity expected under the present experimental conditions. Antiviral activities relative to that of human EDN are shown. For antibacterial activity, + and − indicate activity detected or undetected. MY, million year (7, 22–24, 26, 27, 31, 33).

Figure 2

The phylogenetic tree used for the inference of ancestral sequences. Sequences determined in this study are marked with *.

Figure 3

The present-day and ancestral sequences of EDN and ECP. Only the mature peptides (without signal sequences) are shown. The two important sites discussed in the text are boxed. The three major catalytic residues and eight structural cysteines are marked with solid and open arrows, respectively. The ancestral amino acids with <50% posterior probabilities are underlined. The parsimony alternative ancestral states are P, or G, or L at site 21; R, or T, or A at site 65; and S or D at site 99. Experiments show that these three sites are unimportant to the RNase activity. “?” in ancestor B indicates equally parsimonious inferences of Arg or Val. CE macaque, crab-eating macaque; PT macaque, pig-tailed macaque; SQ monkey, squirrel monkey; CT tamarin, cotton-top tamarin; RB tamarin, red-bellied tamarin.

Figure 4

Crystal structures of human EDN and ancestral protein A, showing the physical interaction between residues at positions 64 and 132. The protein backbones are shown with the spacefill presentation of the residues at the two important sites as well as that of the catalytic His-129 (in purple). The structure of the ancestral protein A was modeled according to the human EDN structure (PDB Id: 1HI2) by SWISS-MODEL (53). Both structures are depicted with the RASMOL program (54).

Figure 5

Complementary substitutions in the evolutionary enhancement of RNase activity in EDN. (A) The present-day and ancestral amino acids at the two critical sites. (B) Evolutionary scenarios of the complementary substitutions. The height of and the number on each arrow show the relative RNase activity of the protein.