Epistasis in protein evolution

Abstract

The structure, function, and evolution of proteins depend on physical and genetic interactions among amino acids. Recent studies have used new strategies to explore the prevalence, biochemical mechanisms, and evolutionary implications of these interactions-called epistasis-within proteins. Here we describe an emerging picture of pervasive epistasis in which the physical and biological effects of mutations change over the course of evolution in a lineage-specific fashion. Epistasis can restrict the trajectories available to an evolving protein or open new paths to sequences and functions that would otherwise have been inaccessible. We describe two broad classes of epistatic interactions, which arise from different physical mechanisms and have different effects on evolutionary processes. Specific epistasis-in which one mutation influences the phenotypic effect of few other mutations-is caused by direct and indirect physical interactions between mutations, which nonadditively change the protein's physical properties, such as conformation, stability, or affinity for ligands. In contrast, nonspecific epistasis describes mutations that modify the effect of many others; these typically behave additively with respect to the physical properties of a protein but exhibit epistasis because of a nonlinear relationship between the physical properties and their biological effects, such as function or fitness. Both types of interaction are rampant, but specific epistasis has stronger effects on the rate and outcomes of evolution, because it imposes stricter constraints and modulates evolutionary potential more dramatically; it therefore makes evolution more contingent on low-probability historical events and leaves stronger marks on the sequences, structures, and functions of protein families.

Keywords: ancestral sequence reconstruction; deep mutational scanning; epistasis; evolutionary biochemistry; protein evolution; sequence space; sequence-function relationship.

Figure 1

Patterns of epistasis between mutations. (A and B) Mutations a⇒A and b⇒B behave additively (non‐epistatically) with respect to the measured phenotype (e.g., stability, fitness): the phenotypic effect of a state at one site is independent of the state of the other. (C and D) The two mutations exhibit negative epistasis: the double mutant AB has a lower measured phenotype than would be expected from the effects of A and B alone, regardless of the direction of the effect.1 (E and F) The two mutations exhibit positive epistasis: the double mutant AB has a greater phenotype than would be expected from the effects of A and B alone. (G) The two mutations exhibit negative sign epistasis: the sign of the phenotypic effect of state B depends on the state at site A. (H) The two mutations exhibit reciprocal sign epistasis: the sign of the phenotypic effect of either mutation changes depends on the state at the other.

Figure 2

Evidence for epistasis in extant sequence data. (A) A mutation from F to V at a given site is known to cause disease in humans, but V is observed as the wild‐type state in mouse, implying permissive mutations in mouse or restrictive mutations in human. (B) Amino acid usage for a site in a given clade (u_a or u_b) only represents a fraction of the total amino acid usage observed for the site over its long‐term evolution (u_T), suggesting different epistatic constraints between clades. (C) Phylogenetic analysis is used to infer historical amino acid substitutions (indicated by vertical bars; additional sequences used to polarize changes leading to bottom two sequences are not shown). The number of paired substitutions to the same amino acid state (convergent substitutions, N_c) decreases with increasing evolutionary distance, relative to the number of paired substitutions to different amino acid states (divergent substitutions, N_d), suggesting lineage‐specific constraints on acceptable states. (D) Pairs of sites that exhibit significant covariation correspond to protein structural contacts.

Figure 3

Mechanisms of epistasis and their evolutionary implications. Biological properties (e.g., function, fitness) depend on the physical properties of protein molecules (e.g., stability, solubility, affinity for ligand), which in turn depend on the peptide sequence. Epistasis is defined as nonadditivity somewhere in the mapping from sequence to biological properties. Specific epistasis causes nonadditivity in the mapping from sequence to physical properties because of physical interactions between sites. Nonspecific epistasis arises from an intrinsic nonlinear relationship between physical and biological properties. Specific permissive mutations enable fewer mutations than nonspecific permissive mutations and therefore have a less dramatic impact on protein evolvability. Similarly, mutations that require a specific permissive mutation to be tolerated have fewer possible permissive mutations than a mutation that can be enabled through a nonspecific effect. This causes specific epistasis to underlie stronger historical contingency, lower reversibility, and stronger long‐term evolutionary constraints.

Figure 4

Examples of specific and nonspecific epistasis. (A) The relationship between stability and function is often modeled by a sigmoidal function as expected due to the nonlinear relation between stability and the fraction of protein folded at a given temperature. This relationship is projected onto the y‐ and z‐axes of the mutational reaction coordinates of (B) and (C) as a gradient from white (functional) to red (nonfunctional). (B) A graphical example of specific epistasis. The red mutation in the parental background is destabilizing, resulting in a large functional defect. However, a blue mutation, which by itself does not alter stability, interacts with the red mutation to reduce its effect on stability and, in turn, on function. (C) An example of nonspecific epistasis. The red mutation in the parental background is destabilizing, resulting in a large functional defect. The blue mutation is stabilizing and by itself has little impact on function. However, in this stability‐buffered background, the red mutation (the destabilizing effect of which is unchanged) can occur with no functional defect.