Alan Wardroper
Workshop: Introduction to Molecular Structure 
Jan 13-16, 2004

Molecule: Avian Oxy Haemoglobin (bar-headed goose, Anser indicus)

PDB Filename: 1a4f.pdb

X-ray Diffraction  Resolution: 2.00 Å

Ref: Zhang J, Hua Z, Tame JR, Lu G, Zhang R, Gu X.  The crystal structure of a high oxygen affinity species of haemoglobin (bar-headed goose haemoglobin in the oxy form). J. Mol. Biol. 1996 Jan 26; 255(3): 484-93.

General

Molecule: Avian Oxy Haemoglobin

Source: Anser indicus. Bar-headed goose

PDB Filename: 1a4f.pdb (Zhang et al., 1996. The crystal structure of a high oxygen affinity species of haemoglobin (bar-headed goose haemoglobin in the oxy form).

Introduction

The bar-headed goose Anser indicus has markedly higher tolerance for low oxygen pressures than related lowland species, such as the greylag goose A. anser, which is reflected in the significantly higher oxygen affinity of its haemoglobin (Hb), allowing it to survive at higher altitudes. There are only four amino acid differences between the Hbs of these two species. However, substitution of Pro to Ala α119 in bar-headed goose Hb reduces contact between the α₁ and β₁ subunits as compared with A. anser, which confers increased oxygen affinity by destabilising the deoxy T state of the protein as a result of the relaxation of the α₁β₁ interface. The crystal structure of A. indicus oxy-haemoglobin was solved by X-ray diffraction at a resolution of 2 Å (PDB code, 1a4f; Zhang et al., 1996) and is described here. The structure is also briefly compared and contrasted with that of the deoxy T state (PDB code, 1hv4).

The bar-headed goose haemoglobin molecule shows the same general overall structure as other Hbs characterised to date, consisting of a tetramer of two α- and two β- chains of 141 and 146 residues, respectively, bound to each other noncovalently. The structure is comprised primarily of antiparallel helices (mostly α-helices) linked by short β-turns with no β-sheets (Fig. 1).

Figure 1

Cartoon showing the tetrameric structure of Avian Haemoglobin

The molecule is characterised according to the SCOP classification as having a 6-helix, folded leaf, partly opened core belonging to the globin-like superfamily. Overall, the structure consists of 68.25% α-helix (i→i+4) and 6.775% 3₁₀-helix (i→i+3) by sequence across all chains, as listed in Table 1. There are also three short right-handed 3₁₀ helices, characterised by tighter coiling with an i->i+3 Hydrogen bonding topology. The helices are connected by loops and turns, and the molecule has no beta strands or disulfide bonds. The unstructured loop regions lie mostly on the outside of the molecule, exposed to the surrounding solvent, while the solvent-exposed helices are amphipathic, with hydrophilic polar side chains exposed on the external face – (oxygen and nitrogen are present on the external surface at high frequency – and hydrophobic non-polar side chains facing into the hydrophobic core (Fig. 2 A & B). Figure 7 shows Ramachandran plots indicating the mainly helical secondary structure in both α- and β-chains. Residues βAla119 and αLeu2 have unusual ψ and φ bond torsion angles as indicated in the plots.

Figure 2

Surface α-helices are amphipathic

A) External surface of α-helix

A) Side view of external face of α-helix showing polar side chains exposed to the solvent.

B) End view showing inner (left) and outer (right) faces

B) The other, internal face presents non-polar side chains into the hydrophobic core of the molecule, as shown in this end view: left, internal (hydrophobic); right, external (hydrophilic).

The structure of the avian Hb is similar to that of human HbA (1hhO; Shaanan, 1983), but differences in intersubunit contacts due to the single Pro→Ala mutation described above result in slightly altered packing of the tetramer. In oxy Hb of Anser indicus, the αβ dimers are rotated by an angle of about 4° as compared with human oxy Hb. This unique quaternary structure, designated the R_H state, may be correlated with the increased oxygen affinity of bar-headed goose Hb described above. In the native folded state, the N- and C-termini (Val/Arg and Val/His for α- and β-chains, respectively) of each chain are localized in close proximity to each other and form a strongly positively charged groove at the entrance to the central cavity. The distribution of positive charges around this highly conformationally ordered region suggests that this is the binding site for inositol pentaphosphate in the T state, which binds avian Hb and is required for function in the same way as 2,3-diphosphoglycerate binds and regulates oxygen affinity of mammalian Hb (Rollerma & Bauer, 1979; Arnone, 1972).

Topology 

The α-chain (141 residues), consisting of seven α-helices and one 3₁₀ helix linked in antiparallel orientation mostly by β-turns, has the following topology: HTHGTTHTHHTHTHTT (H=helix, T=turn, G=γ-turn). The 146-residue β-chain consisting of seven α- and two 3₁₀-helices, has the following topology: HTHGHHTHHGHT (Tables 1 & 2). Helix 3 in each chain is a right-handed 3₁₀ helix (α- and β-chains, residues 37-42 and 36-42, respectively). The β-chain also has an additional short 3₁₀-helix from residues 43-45 (Fig. 3, Tables 1 & 2).

 

Figure 3

PDB secondary structure cartoon

PDB secondary structure cartoon showing the 7 helices in the A chain and 8 in the B chain.

Generated by STING Millennium Suite

Active sites

There are four active sites in the tetrameric molecule, one per chain, each of which binds a haem group containing one Fe⁺⁺ atom (which is oxidised in 1a4f) (Fig. 4). The key residues for haem group binding are outlined in Table 3. In both α- and β-chains, the haem group is coordinated between two basic His residues and a Lys residue. The proximal His residues, His87 and His92 in the α- and β-chain, respectively, make contact with the haem group Fe⁺⁺ ion, forming a ‘picket fence’ to stabilise the ferrous form of the iron porphyrin ring necessary for reversible binding of O₂ for extended periods (Collman et al., 1975).

In the β-chain, Lys66 forms a Hydrogen bond with the haem group via βLys66Nζ→Haem Ala65 CβH over a distance of 3.294Å. The equivalent residue in the α-chain, Lys61, may also interact with the haem group, although it is located at the greater distance of 3.57Å and no H-bond was detected in the model.

Figures 5A and B show the relative positions of the distal (αHis58; βHis63) and proximal (βHis92; αHis87) His residues, βLys66, and the haem group in Hb the oxy R state. Figure 5B shows the Van der Waal’s surfaces of the haem group and each of the three key residues. βLys66Nζ can be seen to form an H-bond with the haem group Ala65 CβH (Figs. 5A & B).

Figure 5

Haem binding cleft (β-chain) showing contact with His92

A) His92 makes contact with Haem Fe	B) Van der Waal’s radii of contact area

The proximal His residue (βHis92, αHis87) stabilises the Fe⁺⁺ ion in the iron porphyrin ring of the haem group in R state haemoglobin. Figure 5A shows the locations of the basic proximal (βHis92) and distal (βHis63) His residues relative to the haem group. Figure 5B shows the Van der Waal’s surfaces of the haeme group and the three key residues. βLys66Nζ forms an H-bond with the haem group Ala65 CβH.

(Images generated using RasMol)

The hydrophilic propanoate groups of the haem group face the water at the surface of the protein, while the hydrophobic portions of the ring are buried among the hydrophobic amino acids of the protein chains.  Figure 6 shows H₂O molecules coordinated with the haem groups bound to the α- and β-chains.  As can be seen in the figure, the α-chain haem has two coordinated water molecules, while that in the B chain has only 1, and they are located in different positions relative to the porphyrin ring structure (which in itself is altered between the two states, as described in the next section).  In addition, the haem groups can be seen to lie in different planes across the chains, with a difference in angle of ~36° between α₁ and β₁  (Fig. 6).

Figure 6

Water molecules coordinated with haem groups

The hydrophilic propanoate groups of the haem group face the solvent at the surface of the protein, while the hydrophobic portions of the ring are buried among the hydrophobic amino acids of the protein chains. The α-chain haem carries two coordinated water molecules, while that of the β-chain has only 1, and they are located in different positions relative to the porphyrin ring structure.

Fig. 7

Ramachandran Plots

α-Chain	β-Chain

Plotted using STING

Ramachandran plots indicating the mainly helical secondary structure in both α- and β-chains.

βAla119 and αLeu2 have unusual torsion angles:        

βAla119:                PHI, 47.87; PSI, -132.55

αLeu2:                    PHI, 25.75; PSI, 113.78

Comparison between T (tense) and R (relaxed) states 

Comparison of the oxy-Hb structure (1a4h) with the deoxy structure (1hv4) for the same species revealed differences in both the overall structure and the local conformation of the haem group between the two states. In the oxy R form, the Fe atom is lifted up and through the haem ring structure orthologous to the porphyrin plane, which translates throughout the ring causing its shape to change. The change in oxidation state of the haem group also translates to global shifts in relative orientation of domains throughout the molecule. While the distance between βHis63 and the ferrous iron atom changes relatively little with oxidation state [βHis 63↔Fe: Oxy (1a4f), 4.508Å; Deoxy (1hv4), 4.23Å], βHis92 is brought into closer proximity with the haem Fe on O₂ binding [βHis92Oxy↔Fe: Oxy (1a4f), 3.046 Å, Deoxy (1hv4), 2.32Å]. As the Fe atom and βHis63 remain closely associated, this movement of the Fe atom on oxidation pulls the His residue out of its T state position, which is translated to a shift in the entire protein chain into the R state.  These changes are transmitted throughout the protein, ultimately resulting in the marked shift in shape that alters the binding strength of the neighbouring sites. Clear differences in overall structure can be seen in Figure 8, which shows only the β-chains of both oxy and deoxy forms.

Figure 8

Stereo views showing differences in relative positions of haem Fe, βHis63, and βHis92 between T and R states

	Oxy (R) Haemoglobin (1a4f)
	Deoxy (T) Haemoglobin (1hv4)

The figures show stereo views of the haem binding pocket of A. indicus haemoglobin in both R (oxy; top) and T (deoxy; bottom) states.

Note the differences in relative positions of the Fe atom and the haem group. Note also the changes in position and orientation of the proximal and distal His residues as well the shift in position of the auxiliary Lys66 residue between T and R states.

As can be seen from Tables 4 and 5, the inter-atom bond lengths deviate much less from the overall means in Hb in the deoxy state as compared with the oxy state. Although the difference in resolution of the structures will affect these values (1a4f, 2.00 Å; 1hv4, 2.80 Å), these differences reflect the conformational changes known to occur in Hb on changing between oxidation states (Baldwin & Chothia, 1979). The conformational changes that occur on binding of oxygen to the haem Fe are communicated to the interfaces between the subunits, breaking the salt links between the chains leading to a change from the T to the R state on oxidation. Thus, in the deoxy- T, or ‘tense’, conformation the quaternary structure is markedly more constrained than in the R, or ‘relaxed’, conformation. This is especially noticeable at the C-termini of each chain, which have almost full rotational freedom in the R state but are highly constrained by electrostatic interactions in the T state.

Conclusions

The haemoglobin of the bar-headed goose Anser indicus is highly similar to mammalian Hbs as well as those of other avian species both structurally and in its amino acid sequence. The unusually high oxygen affinity of the Hb of this species represents an adaptation to high altitudes, and appears to be due to a single amino acid substitution of the bulky cyclic imino acid residue Pro to the small aliphatic residue Ala at position 119 in the α-chain, removing a hydrophobic contact between the α₁ and β₁ subunits, resulting in a loosening of the contact between the subunits as compared with the Hb from the related lowland species A. anser.  This confers increased oxygen affinity by destabilising the deoxy T state of the protein as a result of the reduced steric hindrance at the α₁β₁ interface.

References

1. Arnone A. X-ray diffraction study of binding of 2,3-diphosphoglycerate to human deoxyhaemoglobin. Nature. 1972 May 19; 237(5351): 146-9.
2. Baldwin J, Chothia C. Haemoglobin: the structural changes related to ligand binding and its allosteric mechanism. J Mol Biol. 1979 Apr 5; 129(2): 175-220.
3. Collman JP, Gagne RR, Reed CA, Halbert TR, Lang G, Robinson WT. "Picket fence porphyrins." Synthetic models for oxygen binding hemoproteins. J Am Chem Soc. 1975 Mar 19; 97(6): 1427-39.
4. Liang, Y, Hua, Z, Liang, X, Xu, Q, Lu, G. The Crystal Structure of Bar-Headed Goose Hemoglobin in Deoxy Form: The Allosteric Mechanism of a Hemoglobin Species with High Oxygen Affinity. J.Mol.Biol. 313 pp. 123 (2001)
5. Rollema HS, Bauer C. The interaction of inositol pentaphosphate with the hemoglobins of highland and lowland geese. J Biol Chem. 1979 Dec 10; 254(23): 12038-43.
6. Shaanan B. Structure of human oxyhaemoglobin at 2.1 Å resolution. J Mol Biol. 1983 Nov 25; 171(1): 31-59.
7. Zhang J, Hua Z, Tame JR, Lu G, Zhang R, Gu X.  The crystal structure of a high oxygen affinity species of haemoglobin (bar-headed goose haemoglobin in the oxy form). J. Mol. Biol. 1996 Jan 26; 255(3): 484-93.

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Tables

Table 1::Comparison of structural features between Oxy/Deoxy Hb

Table 2::ProMotif Summary

Table 3::Key Residues for Haem Binding

Table 4::Oxy Hb Chain Bond Lengths

Table 5::Deoxy Hb Chain Bond Lengths

Table 6::Oxy Haemoglobin Helix Details

Table 7A::List of putative hydrogen bonds formed by LYS 61 (α-chain) in PDB entry 1A4F

Table 7B::List of putative hydrogen bonds formed by LYS 66 (β-chain) in PDB entry 1A4F

Table 7C::List of putative hydrogen bonds formed by HIS 58 (a-chain) in PDB entry 1A4F

Table 7D::List of putative hydrogen bonds formed by HIS 87 (α-chain) in PDB entry 1A4F

Table 8A::List of putative hydrogen bonds formed by HIS 63 (β-chain) in PDB entry 1A4F

Table 8B::List of putative hydrogen bonds formed by HIS 92 (β-chain) in PDB entry 1A4F

Table 9::PROCHECK summary for 1a4f