Unique interactions present in highland hemoglobin.

<p>Locations of unique interactions. Chain A (also referred as α1) is colored in cyan, and chain B (β1) and chain D (β2) are colored in white. The key residues are shown as a stick model, and the circles highlight the locations of identified unique interactions.</p

Interacting amino acids of the 2 tetramers.

<p>Interacting amino acids of the 2 tetramers.</p

Sequence comparisons and differences in the α subunit main chain coordinates between highland and lowland hemoglobin.

<p>(A) Sequence alignment of deer mouse highland (H) and lowland (L) hemoglobin. The substitutions are highlighted in blue. The sequences were obtained from the deposited structures as follows: highland deer mouse hemoglobin (PDB ID: 5KER), lowland deer mouse hemoglobin (4H2L), human hemoglobin (2DN1). (B) Orientation of two tetrameric hemoglobin molecules in an asymmetric unit. The α subunits are colored in cyan, and the β subunits are colored in gray. Heme in each subunit is shown as a red stick model, and the active site without ligand assignment is indicated with a black circle. (C) Structural difference in main chain folding with an emphasis on αHis45 coordinates. Tetrameric highland hemoglobin (5KER; cyan) and lowland hemoglobin (4H2L, pink) are superimposed by PyMOL. The residues around the active site and the nearby loop region are shown in the cartoon model. Key residues and helices containing these residues in the α subunit (chain A) are shown in the stick model and the cartoon model.</p

Alteration of the α₁β₂/α₂β₁ subunit interface contributes to the increased hemoglobin-oxygen affinity of high-altitude deer mice

<div><p>Background</p><p>Deer mice (<i>Peromyscus maniculatus</i>) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant.</p><p>Results</p><p>Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules.</p><p>Conclusions</p><p>The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α₁β₂/α₂β₁ intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.</p></div

Data collection and refinement statistics of the type S crystal.

<p>Data collection and refinement statistics of the type S crystal.</p

Difference in the α₁β₂ switching region interaction between highland and lowland hemoglobin.

<p>The α subunit is shown in the cartoon, and the key residues are represented as a stick model. The residues unique to highland and lowland hemoglobin are labeled in cyan and pink, respectively. The highland hemoglobin α₁ subunit is colored in cyan, and its β₂ subunit is colored in gray. Both subunits from the lowland hemoglobin are colored in pink. The right side boxes indicate differences in the α₁β₂/α₂β₁ interface coordinates, particularly the β₂His 97 imidazole ring orientation relative to the α₁ residues.</p

Molecular basis of hemoglobin adaptation in the high-flying bar-headed goose

<div><p>During the adaptive evolution of a particular trait, some selectively fixed mutations may be directly causative and others may be purely compensatory. The relative contribution of these two classes of mutation to adaptive phenotypic evolution depends on the form and prevalence of mutational pleiotropy. To investigate the nature of adaptive substitutions and their pleiotropic effects, we used a protein engineering approach to characterize the molecular basis of hemoglobin (Hb) adaptation in the high-flying bar-headed goose (<i>Anser indicus</i>), a hypoxia-tolerant species renowned for its trans-Himalayan migratory flights. To test the effects of observed substitutions on evolutionarily relevant genetic backgrounds, we synthesized all possible genotypic intermediates in the line of descent connecting the wildtype bar-headed goose genotype with the most recent common ancestor of bar-headed goose and its lowland relatives. Site-directed mutagenesis experiments revealed one major-effect mutation that significantly increased Hb-O₂ affinity on all possible genetic backgrounds. Two other mutations exhibited smaller average effect sizes and less additivity across backgrounds. One of the latter mutations produced a concomitant increase in the autoxidation rate, a deleterious side-effect that was fully compensated by a second-site mutation at a spatially proximal residue. The experiments revealed three key insights: (<i>i</i>) subtle, localized structural changes can produce large functional effects; (<i>ii</i>) relative effect sizes of function-altering mutations may depend on the sequential order in which they occur; and (<i>iii</i>) compensation of deleterious pleiotropic effects may play an important role in the adaptive evolution of protein function.</p></div

Compensatory interaction between spatially proximal α-chain residues in bar-headed goose Hb.

<p>The mutation Aα63V produces a >2-fold increase in autoxidation rate (<i>k</i>_auto; ± 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 (<i>A</i> and <i>B</i>) in which mutations at both sites are tested individually and in pairwise combination.</p

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 <i>Anser</i> species.

<p>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.</p

Bar-headed goose evolved an increased Hb-O₂ affinity relative to greylag goose and their reconstructed ancestor, AncAnser.

<p>Triangulated comparisons of purified rHbs involved diffusion-chamber measurements of O₂-equilibria (<i>A</i>) and stopped-flow measurements of O₂ dissociation kinetics (<i>B</i>). O₂-affinities (<i>P</i>₅₀, torr; ± 1 SEM) and dissociation rates (<i>k</i>_off, 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 (<i>P</i><0.05).</p