Structure-function relationship in a variant hemoglobin: a combined computational-experimental approach

Abstract

Our study examines the functional and structural effects of amino acid substitution in the distal side of beta-chains of human Hb Duarte (alpha(2)beta(2)(62Ala-->Pro)). We have compared the functional properties of the purified Hb Duarte with those of HbA, and through proton NMR and molecular dynamics simulations we have investigated their tertiary and quaternary structures. The variant exhibits an increased oxygen affinity with a normal Hill coefficient and Bohr effect. The abnormal function of Hb Duarte is attributed to the presence of a proline residue at the beta62 position, since the functional properties of another Hb variant in the same position, Hb J-Europa (beta(62Ala-->Asp)), have been described as normal. Thereafter (1)H-NMR studies have shown that the beta62 Ala-->Pro substitution causes structural modifications of the tertiary structure of the beta globins, leaving the quaternary structure unaltered. These results have been confirmed by extensive all-atom molecular dynamics simulations. All these findings lead to the conclusion that the beta62 Ala-->Pro substitution produces a destabilization of the E-helix extending downward to the CD corner. Particularly, a cavity near the distal histidine of the beta-chains, connecting the heme pocket to the solvent, is affected, altering the functional properties of the protein molecule.

FIGURE 1

Deoxy hemoglobin with the α1 (red), α2 (orange), β1 (blue), and β2 (gray) subunits. Heme groups are evidenced via licorice representation, and the substituted ALA residues are in yellow. This figure as well as all the structural figures were obtained with the visualization program VMD (59).

FIGURE 2

(a) IEF of total hemolysate (line 1), HbA (line 2), and Hb components purified by IEC (lines 3 and 4); (b) RP-HPLC of the globin chains of HbA and purified Hb Duarte.

FIGURE 3

Effect of pH on the oxygen affinity (log p₅₀) and cooperativity (n₅₀) at (a) 25°C, and (b) 37°C of HbA (circles) and purified Hb Duarte (triangles) in the absence (open symbols) and in the presence (solid symbols) of 5 mM 2,3-DPG. Experimental conditions: 100 mM Bis-Tris/Tris + 100 mM NaCl. O₂ pressure is expressed in torr.

FIGURE 4

Downfield ring current-shifted resonances of HbA and Hb Duarte in the CO form.

FIGURE 5

Downfield hyperfined-shifted N_δH of proximal histidines resonances of HbA and Hb Duarte in the deoxy form.

FIGURE 6

Hyperfine shifted proton resonances of HbA and Hb Duarte in the deoxy form collected at several temperatures.

FIGURE 7

Hyperfine shifted and exchangeable proton resonances of HbA and Hb Duarte in the deoxy form collected at several temperatures.

FIGURE 8

Exchangeable proton resonances of HbA and Hb Duarte in the CO form.

FIGURE 9

Xrms of deoxy HbA: (a) tetramer, (b)–(e) β2, α2, β1, and α1 chains, respectively. The black curves refer to the deviations, the gray curves to the absolute deviations.

FIGURE 10

Xrms of deoxy HbA (black curves) and mutated (gray curves) hemoglobins: (a) tetramer, (b)–(e) β2, α2, β1, and α1 chains, respectively.

FIGURE 11

Probability distributions of the distances in the switch region of the deoxy Hbs: (upper panel) Asp-94–Trp-37 at the α1β2 interface; (central panel) His-103–Gln-131 at the α1β1 interface; (lower panel) Tyr-42–Asp-99 at the α1β2 interface. Solid and dotted curves are for HbA and Duarte variants, respectively. The dash-dot lines indicate the corresponding distances in the x-ray structures.

FIGURE 12

Probability distribution of the distances N_δ(His F8)-CO(Leu F4) (black curves) and N_bckbn(His F8)-CO(LEU F4) (gray curves) for the deoxy Hbs. The solid and dotted curves refer to data for HbA and Hb Duarte, respectively. In the inset, the N ions and the leucine residue involved in the formation of H-bonds are shown.

FIGURE 13

Probability distribution of the angles N_δ(His F8)-CO(Leu F4) (black curves) and N_bckbn(His F8)-CO(Leu F4) (gray curves) for the deoxy Hbs. The solid and dotted curves refer to data for HbA and Hb Duarte, respectively.

FIGURE 14

Distribution of the distance CG(1-2)(ValE11)-Fe (see inset) in the β- (upper panel) and α- (lower panel) chains for deoxy HbA (solid lines) and deoxy Hb Duarte (dashed lines) during the simulation.

FIGURE 15

Helix section E1–E6 in Hb Duarte (blue tube) and HbA (orange tube), respectively, after 6 and 9 ns of MD simulation.

FIGURE 16

Distribution of the distance CZ(PheCD4)-CG(HisE7) (see inset) in the β- (upper panel) and α- (lower panel) chains for deoxy HbA (solid lines) and deoxy Hb Duarte (dashed lines) during the simulation. The vertical dotted lines refer to the values extracted from the x-ray structure.

FIGURE 17

Cavity position in hemoglobin (see Table 2). The white spheres refer to the position of the center of mass of the cavities.