Structure of liganded T-state haemoglobin from cat (Felis silvestris catus), a low oxygen-affinity species, in two different crystal forms

Abstract

Haemoglobin (Hb) is an iron-containing metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. Hb is in equilibrium between low-affinity tense (T) and high-affinity relaxed (R) states associated with its unliganded and liganded forms, respectively. Mammalian species can be classified into two groups on the basis of whether they express `high' or `low' oxygen-affinity Hbs. Although Hbs from the former group have been studied extensively, a more limited number of structural studies have been performed for low oxygen-affinity Hbs. Here, the crystal structure of low oxygen-affinity cat methaemoglobin (metHb) has been solved at 2.0 and 2.4 Å resolution in two different crystal forms. Even though both structures are fully liganded, they unusually adopt a T-state-like quaternary conformation but with several localized R-like tertiary-structural and quaternary-structural features. The study provides atomic-level insights into the ligand-binding properties of this Hb, including its low cooperativity, blunt response to allosteric effectors and low affinity for oxygen, as well as further contributing to the mechanism underlying Hb allostery.

The structure of haemoglobin bound to the haemoglobin receptor IsdH fromStaphylococcus aureusshows disruption of the native α-globin haem pocket

“…Hydrogen bonds and salt bridges at the IsdH N2N3 -metHb interface are shown in Table 2. Symmetry-related dimers form a Hb tetramer in the crystal; however, the relative positioning of dimers does not correspond to the canonical T (low O 2 affinity) or R (high O 2 affinity) quaternary structures of Hb (Perutz et al, 1960;Muirhead & Perutz, 1963) and is more similar to two recently determined structures of fully liganded Hbs with a T-like quaternary structure (Safo et al, 2013;Balasubramanian et al, 2014; discussed further below).…”

“…The iron-iron distance between haems of IsdH N2N3 -metHb (Table 4; 38.3 Å ) also indicated closer similarity to the T state (39.5 Å ) than to the R state (34.9 Å ). Using the same criteria (Tables 3 and 4), two other Hb structures in the PDB show much greater levels of similarity to IsdH N2N3 -metHb, namely a human Hb variant comprising embryonic chain and chain carrying the sicklecell mutation ( S ) crystallized in the fully CO-ligated form (Safo et al, 2013) and metHb from cat (Felis silvestris catus; Balasubramanian et al, 2014). Both the Hb 2 2 S and cat metHb structures exhibit an unusual T state-like quaternary structure with several local tertiary and quaternary features characteristic of the R state (Safo et al, 2013;Balasubramanian et al, 2014).…”

“…† The T-state and R-state reference structures are human deoxy-Hb and HbCO fromPark et al (2006), respectively, the R2 structure is fromSilva et al (1992), the RR2 and R3 structures are fromSafo & Abraham (2005), the human variant Hb comprising Hb and S (carrying the sickle-cell mutation) is fromSafo et al (2013) and the low-affinity Hb from F. silvestris catus is fromBalasubramanian et al (2014). ‡ Comparison with the 4xs0 structure after superposing 11 dimers using residues of the BGH frame.…”

Genetically based low oxygen affinities of felid hemoglobins: lack of biochemical adaptation to high-altitude hypoxia in the snow leopard

“…This interpretation agrees with the minor role of the β-subunit Nterminus in DPG binding (Richard et al, 1993) compared with the original model for DPG binding proposed by Arnone (1972). In addition to impairing DPG binding, the β2His→Phe substitution that exchanges a basic residue with an apolar one also contributes to the reduced intrinsic O 2 affinity typical of feline Hb isoforms by moving the β-chain N-termini towards the center of the Hb molecule, thereby stabilizing the T-state conformation (Perutz et al, 1993;Safo and Abraham, 2001) even when the heme is fully ligated, as found for domestic cat Hb (Balasubramanian et al, 2014). Similarly, the low O 2 affinity and DPG insensitivity of bovine Hb is also attributable to amino acid substitutions in the β-subunit N-termini (β1Val deleted and β2His→Met), which stabilize the low-affinity T state and prevent DPG access to the central cavity (Perutz et al, 1993;Safo and Abraham, 2001).…”

Molecular insights of an avian species with low oxygen affinity, the crystal structure of duck methemoglobin

“…The column was preequilibrated with water and the hemolyzed sample was gently loaded on the column. Initially the column was washed with water to elute the unbound proteins, thereafter eluted with a gradient of NaCl salt solution, ranging from 0.1 M to 1 M, by stepwise increasing of 0.1 M. The bound hemoglobin was eluted at 0.1 M NaCl gradient elution and collected at a rate of 3 ml/min [10,[21][22][23][24][25][26][27][28]. The homogeneity of hemoglobin samples was analyzed by using SDS-PAGE [29].…”