Intermolecular interactions drive protein adaptive and co-adaptive evolution at both species and population levels

Proteins are the building blocks for almost all the functions in cells. Understanding the molecular evolution of proteins and the forces that shape protein evolution is essential in understanding the basis of function and evolution. Previous studies have shown that adaptation frequently occurs at the protein surface, such as in genes involved in host-pathogen interactions. However, it remains unclear whether adaptive sites are distributed randomly or at regions associated with particular structural or functional characteristics across the genome, since many proteins lack structural or functional annotations. Here, we seek to tackle this question by combining large-scale bioinformatic prediction, structural analysis, phylogenetic inference, and population genomic analysis of Drosophila protein-coding genes. We found that protein sequence adaptation is more relevant to function-related rather than structure-related properties. Interestingly, intermolecular interactions contribute significantly to protein adaptation. We further showed that intermolecular interactions, such as physical interactions, may play a role in the co-adaptation of fast-adaptive proteins. We found that strongly differentiated amino acids across geographic regions in protein-coding genes are mostly adaptive, which may contribute to the long-term adaptive evolution. This strongly indicates that a number of adaptive sites tend to be repeatedly mutated and selected in evolution, in the past, present, and maybe future. Our results highlight the important roles of intermolecular interactions and co-adaptation in the adaptive evolution of proteins both at the species and population levels.

Heterogeneity in conservation of multifunctional partner enzymes with meiotic importance, CDK2 kinase and BRCA1 ubiquitin ligase

The evolution of proteins can be accompanied by changes not only to their amino acid sequences, but also their structural and spatial molecular organization. Comparison of the protein conservation within different taxonomic groups (multifunctional, or highly specific) allows to clarify their specificity and the direction of evolution. Two multifunctional enzymes, cyclin-dependent kinase 2 (CDK2) and BRCA1 ubiquitin ligase, that are partners in some mitotic and meiotic processes were investigated in the present work. Two research methods, bioinformatics and immunocytochemical, were combined to examine the conservation levels of the two enzymes. It has been established that CDK2 is a highly conserved protein in different taxonomic lineages of the eukaryotic tree. Immunocytochemically, a conserved CDK2 pattern was revealed in the meiotic autosomes of five rodent species and partially in domestic turkey and clawed frog. Nevertheless, variable CDK2 distribution was detected at the unsynapsed segments of the rodent X chromosomes. BRCA1 was shown to be highly conserved only within certain mammalian taxa. It was also noted that in those rodent nuclei, where BRCA1 specifically binds to antigens, asynaptic regions of sex chromosomes were positive. BRCA1 staining was not always accompanied by specific binding, and a high nonspecificity in the nucleoplasm was observed. Thus, the studies revealed different conservation of the two enzymes at the level of protein structure as well as at the level of chromosome behavior. This suggests variable rates of evolution due to both size and configuration of the protein molecules and their multifunctionality.

TRIAD: a transposition-based approach for gene mutagenesis by random short in-frame insertions and deletions for directed protein evolution

Insertions and deletions (InDels) are among the most frequent changes observed in natural protein evolution, yet their potential has hardly been harnessed in directed evolution experiments. Here we describe the standard protocol for TRIAD (Transposition-based Random Insertion And Deletion mutagenesis), a simple and efficient Mu transposon mutagenesis approach for generating libraries of single InDel variants with one, two or three triplet nucleotide insertions or deletions. This method has recently been employed in three published examples of InDel-based directed evolution of proteins, including a phosphotriesterase, a scFv antibody and an ancestral luciferase.

A mutation-selection model of protein evolution under persistent positive selection

We use first principles of population genetics to model the evolution of proteins under persistent positive selection (PPS). PPS may occur when organisms are subjected to persistent environmental change, during adaptive radiations, or in host-pathogen interactions. Our mutation-selection model indicates protein evolution under PPS is an irreversible Markov process, and thus proteins under PPS show a strongly asymmetrical distribution of selection coefficients among amino acid substitutions. Our model shows the criteria ω > 1 (where ω is the ratio of non-synonymous over synonymous codon substitution rates) to detect positive selection is conservative and indeed arbitrary, because in real proteins many mutations are highly deleterious and are removed by selection even at positively-selected sites. We use a penalized-likelihood implementation of our model to successfully detect PPS in plant RuBisCO and influenza HA proteins. By directly estimating selection coefficients at protein sites, our inference procedure bypasses the need for using ω as a surrogate measure of selection and improves our ability to detect molecular adaptation in proteins.

Intermolecular interactions drive protein adaptive and co-adaptive evolution at both species and population levels

Proteins are the building blocks for almost all the functions in cells. Understanding the molecular evolution of proteins and the forces that shape protein evolution is an essential step in understanding the basis of function and evolution. Previous studies have shown that adaptation occurs frequently at the protein surface, such as in genes involved in host-pathogen interactions. However, it remains unclear whether adaptive sites are distributed randomly or at regions that are associated with particular structural or functional characteristics across the genome, since many of the proteins lack structural or functional annotations. Here, we seek to tackle this question by combining large-scale bioinformatic prediction, structural analysis, phylogenetic inference, and population genomic analysis of Drosophila protein-coding genes. By estimating and comparing the rate of adaptive substitutions at protein and residue level, we showed that adaptation is more relevant to function-related rather than structure-related properties. Among the function-related properties, we found that molecular interactions in proteins contribute to adaptive evolution, and putative binding residues exhibit higher rates of adaptation. We observed that physical interactions might play a role in the co-adaptation of fast-adaptive proteins. We found that strongly differentiated amino acids in protein coding genes are mostly adaptive, which may contribute to the long-term adaptive evolution. Our results suggest important roles of intermolecular interactions and co-adaptation in the adaptive evolution of proteins both at the species and population levels.

ANCESTRAL RECONSTRUCTION OF A β-LACTAMASE AND COMPARISON WITH ITS EXTANT PROTEINS

The β-lactamases are proteins of bacterial origin that are characterized by hydrolyzing antibiotics β-lactams, conferring microbial resistance against them. They are a heterogeneous family of proteins very relevant from a health point of view due to the ease they present to acquire resistance to new drugs due to their high capacity for evolution. The in vitro evolution of these proteins has served not only to develop their characterization and improve their knowledge, but as a new line of research that allows to predictively identify residues involved in the acquisition of antibiotic resistance. At the same time, the method of ancestral protein reconstruction has been revealed as a novel and useful tool to understand the evolution of β-lactamases and understand some of their characteristics such as their promiscuity. In this work, a study of ancestral β-lactamases reconstructed from the phylogeny of existing class A β-lactamases has been carried out. Of the four ancestral proteins studied, one has been obtained that is functional and has compared its hydrolytic activity with that of four of its current counterparts against eight β-lactam drugs. This ancestral protein has been shown to have a more generalistic antibiotic activity than any of the current proteins studied. In addition, the active ancestral protein showed more resistance to one of the drugs used than the rest of β-lactamases existing. Finally these results have been discussed and from them it is argued why reconstructed ancestral sequences can be a very attractive starting point when it comes to direct evolution of proteins for obtaining proteins of biotechnological interest.

Structural Heterogeneities of the Ribosome: New Frontiers and Opportunities for Cryo-EM

The extent of ribosomal heterogeneity has caught increasing interest over the past few years, as recent studies have highlighted the presence of structural variations of the ribosome. More precisely, the heterogeneity of the ribosome covers multiple scales, including the dynamical aspects of ribosomal motion at the single particle level, specialization at the cellular and subcellular scale, or evolutionary differences across species. Upon solving the ribosome atomic structure at medium to high resolution, cryogenic electron microscopy (cryo-EM) has enabled investigating all these forms of heterogeneity. In this review, we present some recent advances in quantifying ribosome heterogeneity, with a focus on the conformational and evolutionary variations of the ribosome and their functional implications. These efforts highlight the need for new computational methods and comparative tools, to comprehensively model the continuous conformational transition pathways of the ribosome, as well as its evolution. While developing these methods presents some important challenges, it also provides an opportunity to extend our interpretation and usage of cryo-EM data, which would more generally benefit the study of molecular dynamics and evolution of proteins and other complexes.