Stability-Mediated Epistasis Restricts Accessible Mutational Pathways in the Functional Evolution of Avian Hemoglobin

Abstract

If the fitness effects of amino acid mutations are conditional on genetic background, then mutations can have different effects depending on the sequential order in which they occur during evolutionary transitions in protein function. A key question concerns the fraction of possible mutational pathways connecting alternative functional states that involve transient reductions in fitness. Here we examine the functional effects of multiple amino acid substitutions that contributed to an evolutionary transition in the oxygenation properties of avian hemoglobin (Hb). The set of causative changes included mutations at intradimer interfaces of the Hb tetramer. Replacements at such sites may be especially likely to have epistatic effects on Hb function since residues at intersubunit interfaces are enmeshed in networks of salt bridges and hydrogen bonds between like and unlike subunits; mutational reconfigurations of these atomic contacts can affect allosteric transitions in quaternary structure and the propensity for tetramer-dimer dissociation. We used ancestral protein resurrection in conjunction with a combinatorial protein engineering approach to synthesize genotypes representing the complete set of mutational intermediates in all possible forward pathways that connect functionally distinct ancestral and descendent genotypes. The experiments revealed that 1/2 of all possible forward pathways included mutational intermediates with aberrant functional properties because particular combinations of mutations promoted tetramer-dimer dissociation. The subset of mutational pathways with unstable intermediates may be selectively inaccessible, representing evolutionary roads not taken. The experimental results also demonstrate how epistasis for particular functional properties of proteins may be mediated indirectly by mutational effects on quaternary structural stability.

Keywords: Caprimulgidae; epistasis; fitness landscape; hemoglobin; nightjars; pleiotropy.

Fig. 1

O₂-affinities of HbA and HbD isoforms from high- and low-altitude nightjar species, Hydropsalis longirostris and H. decussata, respectively. (A) Log-transformed P₅₀ values (± 1 SE) for purified HbA isoforms measured in 0.1 M HEPES buffer at pH 7.4, 37 °C, in the absence (stripped) and presence of allosteric effectors ([Cl⁻], 0.1 M; IHP/Hb tetramer ratio, 2.0; [heme], 0.3 mM). (B) P₅₀ values for HbD isoforms (experimental conditions as in A).

Fig. 2

Amino acid substitutions that distinguish the HbA and HbD isoforms of Hydropsalis longirostris and H. decussata and amino acid states at orthologous sites in other New World nightjar species. At each site, derived (nonancestral) amino acids are denoted by red lettering. We used gene-specific phylogeny reconstructions to infer the polarity of character state changes at divergent sites in the α^A-, α^D-, and β^A-globin genes (see supplementary fig. S2, Supplementary Material online).

Fig. 3

Sign epistasis for Hb-O₂ affinity constrains the number of selectively accessible mutational pathways from the ancestral GVAI genotype to the descendent SIVV genotype. (A) O₂-affinities (P₅₀, torr; ± 1 SE) of 16 purified rHbs representing all possible genotypic combinations of bi-allelic variation at the four sites that distinguish GVAI and SIVV. Estimates of P₅₀ for “stripped” Hbs provide measures of intrinsic O₂ affinity, whereas estimates of P₅₀ in the “KCl + IHP” treatment provide measures of Hb-O₂ affinity in the presence of allosteric effectors. At each site, the derived (nonancestral) amino acids are underlined. (B) Trajectories of change in Hb-O₂ affinity (indexed by P_{50(KCl + IHP)}) in each of 24 forward pathways connecting the ancestral GVAI and the descendant SIVV. Dashed horizontal lines depict boundaries of the “neutral corridor” (see text for details). Of the 24 possible forward pathways, 12 included one or more mutational intermediates with values of P_{50(KCl + IHP)} that fell well below the floor of the neutral corridor (trajectories shown in red lines); these putatively inaccessible pathways included intermediates with highly aberrant functional properties (SIAI, SVAV, and SIVI). An additional four pathways passed through the GIAV genotype, which exhibited a marginally significant reduction in P_{50(KCl + IHP)} below the floor of the neutral corridor (trajectories shown in grey). Error bars denote 95% confidence intervals.

Fig. 4

Four-dimensional depiction of sequence space, showing all possible mutational pathways connecting the high-affinity, ancestral GVAI genotype and the low-affinity, quadruple-mutant SIVV genotype (depicted by green and red nodes, respectively). All 16 genotypes are represented as nodes (vertices of the hypercube), with edges connecting genotypes that differ by a single point mutation. Putatively inaccessible pathways that pass through the SIAI, SVAV, and SIVI genotypes are denoted by light grey edges; pathways that pass through the GIAV genotype (which had a marginally significant reduction in P_{50(KCl + IHP)}) are denoted by darker, bluish grey edges; accessible pathways, in which each successive mutational step resulted in an unchanged or reduced Hb-O₂ affinity (increased P₅₀) are denoted by blue edges.

Fig. 5

FPLC gel filtration elution profile for recombinant Hbs (rHbs) representing SIVV, the wild-type genotype of H. decussata, and three rHb mutants (SIAI, SVAV, and SIVI) that exhibited aberrant O₂-binding properties. As discussed in the text, these three rHb mutants exhibited unusually high O₂-affinities in the presence of allosteric effectors (i.e., low values of P_{50(KCL + IHP)}), which suggested that they may have an increased propensity for tetramer–dimer dissociation. Monomeric horse myoglobin (Mb) and tetrameric human Hb were used as standard molecular weight markers. In the plot of elution peak against elution volume for the four rHbs, a shift in the equilibrium towards tetramer–dimer dissociation is indicated by elution volumes shifted towards the Mb value. The gel filtration elution profiles indicate that the three rHbs with suppressed sensitivities to IHP have an increased tendency to dissociate into dimers.

Fig. 6

Amino acid mutations at intradimeric interfaces reduce Hb-O₂ affinity via different structural mechanisms. (A,B) Replacing Ala with Val at α34V results in the gain of two additional intradimer atomic contacts per Hb tetramer, as Vα34 forms a van der Waals contact with Aβ128 in each αβ dimer. This stabilization of α₁β₁/α₂β₂ contacts is predicted to restrict allosteric motion, thereby reducing O₂-affinity by increasing the free energy of the oxygenation-linked T→R transition in quaternary structure. (C,D) Replacing Ile with Val at β112 contributes to a destabilization of the R-state by eliminating two intradimeric van der Waals contacts (Iβ112::Vα107) per Hb tetramer.