Fig 1.
Inferred history of amino acid substitution at five sites that distinguish the major Hb isoforms of the bar-headed goose (Anser indicus) and greylag goose (Anser anser).
(A) Amino acid states at the same sites are shown for 12 other waterfowl species in the subfamily Anserinae. Of the five amino acid substitutions that distinguish the Hbs of A. indicus and A. anser, parsimony indicates that three occurred on the branch leading to A. indicus (αG18S, αA63V, and αP119A) and two occurred on the branch subtending the clade of all Anser species other than A. indicus (βT4S and βD125E). ‘AncAnser’ represents the reconstructed sequence of the A. indicus/A. anser common ancestor, which is also the most recent common ancestor of all extant species in the genus Anser. (B) Triangulated comparisons involving rHbs of bar-headed goose, greylag goose, and their reconstructed ancestor (AncAnser) reveal the polarity of changes in character state. Differences in Hb function between bar-headed goose and AncAnser reflect the net effect of three substitutions (αG18S, αA63V, and αP119A) and differences between greylag goose and AncAnser reflect the net effect of two substitutions (βT4S and βD125E). All possible mutational intermediates connecting AncAnser with each of the two descendent species are shown to the side of each terminal branch (the sequential order of the substitutions is unknown, so the order in which they are shown on each terminal branch is arbitrary).
Fig 2.
Bar-headed goose evolved an increased Hb-O2 affinity relative to greylag goose and their reconstructed ancestor, AncAnser.
Triangulated comparisons of purified rHbs involved diffusion-chamber measurements of O2-equilibria (A) and stopped-flow measurements of O2 dissociation kinetics (B). O2-affinities (P50, torr; ± 1 SEM) and dissociation rates (koff, M-1s-1; ± 1 SEM) of purified rHbs were measured at pH 7.4, 37° C, in the absence (stripped) and presence of allosteric effectors ([Cl-], 0.1 M; [Hepes], 0.1 M; IHP/Hb tetramer ratio = 2.0; [heme], 0.3 mM in equilibrium experiments; [Cl-], 1.65 mM; [Hepes], 200 mM; IHP/Hb tetramer ratio = 2.0; [heme], 5 μM in kinetic experiments). Letters distinguish measured values that are significantly different (P<0.05).
Table 1.
O2 affinities (P50, torr) and anion sensitivities (∆log P50) of rHbs representing bar-headed goose, greylag goose, their reconstructed ancestor (AncAnser), and all possible mutational intermediates connecting AncAnser with each of the two descendant species.
O2 equilibria were measured in 0.1 mM Hepes buffer at pH 7.4 (± 0.01) and 37°C in the absence (stripped) and presence of Cl- ions (0.1 M KCl]) and IHP (at two-fold molar excess over tetrameric Hb). Anion sensitivities are indexed by the difference in log-transformed values of P50 in the presence and absence of Cl- ions (KCl) and IHP. The higher the ∆log P50 value, the higher the sensitivity of Hb-O2 affinity to the presence of a given anion or combination of anions. For the bar-headed goose mutants (all mutational intermediates between wildtype bar-headed goose and AncAnser), three-letter genotype codes denote amino acid states at α18, α63, and α119 (amino acid abbreviations underlined in bold = derived [non-ancestral]). At these same three sites, AncAnser is ‘GAP’ the wildtype genotype of bar-headed goose is ‘SVA’. For the greylag goose mutants (all mutational intermediates between wildtype greylag goose and AncAnser), two-letter genotype codes denote amino acid states at β4 and β125. At these same two sites, AncAnser is ‘TD’ the wildtype genotype of greylag goose is ‘SE’.
Fig 3.
Trajectories of change in intrinsic Hb-O2 affinity (indexed by P50, torr) in each of six possible forward pathways that connect the ancestral ‘AncAnser’ genotype (GAP) and the wildtype genotype of bar-headed goose (SVA).
Derived amino acid states are indicated by red lettering. Error bars denote 95% confidence intervals.
Fig 4.
Structural model showing bar-headed goose Hb in the deoxy state (PDB1hv4), along with locations of each of the three amino substitutions that occurred in the bar-headed goose lineage after divergence from the common ancestor of other Anser species.
The inset graphic shows the environment of the Val α63 residue. When valine replaces the ancestral alanine at this position, the larger volume of the side-chain causes minor steric clashes with two neighboring glycine residues, Gly α25 and Gly α59. The distances between non-hydrogen atoms (depicted by dotted lines) are given in Ǻ.
Fig 5.
Compensatory interaction between spatially proximal α-chain residues in bar-headed goose Hb.
The mutation Aα63V produces a >2-fold increase in autoxidation rate (kauto; ± 1 SEM) on genetic backgrounds with the ancestral Gly at residue position α18. This effect is fully compensated by Gα18S, as indicated by two double-mutant cycles (A and B) in which mutations at both sites are tested individually and in pairwise combination.
Table 2.
Autoxidation rates of rHbs representing bar-headed goose, greylag goose, their reconstructed ancestor (AncAnser), and all possible mutational intermediates connecting AncAnser with each of the two descendant species.
For the bar-headed goose mutants (all mutational intermediates between wildtype bar-headed goose and AncAnser), three-letter genotype codes denote amino acid states at α18, α63, and α119 (amino acid abbreviations underlined in bold = derived [non-ancestral]). At these same three sites, AncAnser is ‘GAP’ the wildtype genotype of bar-headed goose is ‘SVA’. For the greylag goose mutants (all mutational intermediates between wildtype greylag goose and AncAnser), two-letter genotype codes denote amino acid states at β4 and β125. At these same two sites, AncAnser is ‘TD’ the wildtype genotype of greylag goose is ‘SE’.