Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix

Abstract

The deer mouse, Peromyscus maniculatus, exhibits altitude-associated variation in hemoglobin oxygen affinity. To examine the structural basis of this functional variation, the structure of the hemoglobin was solved. Recombinant hemoglobin was expressed in Escherichia coli and was purified by ion-exchange chromatography. Recombinant hemoglobin was crystallized by the hanging-drop vapor-diffusion method using polyethylene glycol as a precipitant. The obtained orthorhombic crystal contained two subunits in the asymmetric unit. The refined structure was interpreted as the aquo-met form. Structural comparisons were performed among hemoglobins from deer mouse, house mouse and human. In contrast to human hemoglobin, deer mouse hemoglobin lacks the hydrogen bond between α1Trp14 in the A helix and α1Thr67 in the E helix owing to the Thr67Ala substitution. In addition, deer mouse hemoglobin has a unique hydrogen bond at the α1β1 interface between residues α1Cys34 and β1Ser128.

Keywords: Peromyscus maniculatus; deer mouse; hemoglobin; oxygen affinity.

Figure 1

Solution UV–Vis spectrum changes in deer mouse hemoglobin during crystallization. 3 µl samples of the protein drops from the crystallization setups were diluted with water and their spectra were recorded.

Figure 2

Sequences and secondary-structure comparisons of deer mouse, house mouse and human hemoglobin. (a) Comparisons between deer mouse hemoglobin and three forms of human hemoglobin. Sequences of deer mouse and human hemoglobin α and β subunits were aligned separately; conserved amino-acid residues are denoted by dots. Secondary structures of three forms of human hemoglobin [the deoxy, oxy and carbonmonoxy (CO) forms] are presented above the human globin sequences and that of deer mouse hemoglobin is presented below the human sequence. H, α-helix; G, three-helix motif; I, five-helix motif; –, residues with no assigned secondary structure. (b) Comparisons between deer mouse hemoglobin and house mouse (Mus musculus) hemoglobin. The sequences of the α and β subunits of deer mouse and house mouse hemoglobin (PDB entry 3hrw) were aligned separately; identical amino-acid residues between the two species are represented as dots in the house mouse sequence. The secondary structure of deer mouse hemoglobin is presented above its sequence and that of house mouse hemoglobin is presented below its sequence.

Figure 3

Superimposed structures of the interactions of the A and E helices in the α subunits of deer mouse hemoglobin and deoxy human hemoglobin. The A, E, F and H helices in the deer mouse hemoglobin (blue) and deoxy human hemoglobin (light pink) α1 subunits are shown. Residues that differ between the two species are shown in color and conserved residues are shown in black. In deoxy human hemoglobin, the A–E helix interaction between α1Thr14 NE1 (marine blue) and α1Thr67 OG1 (red) is represented by a pink dotted line.