Solution structure and dynamics of human hemoglobin in the carbonmonoxy form

Abstract

The solution structure of human adult carbonmonoxy hemoglobin (HbCO A) was refined using stereospecifically assigned methyl groups and residual dipolar couplings based on our previous nuclear magnetic resonance structure. The tertiary structures of individual chains were found to be very similar to the X-ray structures, while the quaternary structures in solution at low salt concentrations resembled the X-ray R structure more than the R2 structure. On the basis of chemical shift perturbation by inositol hexaphosphate (IHP) titration and docking, we identified five possible IHP binding sites in HbCO A. Amide-water proton exchange experiments demonstrated that αThr38 located in the α1β2 interface and several loop regions in both α- and β-chains were dynamic on the subsecond time scale. Side chain methyl dynamics revealed that methyl groups in the α1β2 interface were dynamic, but those in the α1β1 interface were quite rigid on the nanosecond to picosecond and millisecond to microsecond time scales. All the data strongly suggest a dynamic α1β2 interface that allows conformational changes among different forms (like T, R, and R2) easily in solution. Binding of IHP to HbCO A induced small structural and dynamic changes in the α1β2 interface and the regions around the hemes but did not increase the conformational entropy of HbCO A. The binding also caused conformational changes on the millisecond time scale, very likely arising from the relative motion of the α1β1 dimer with respect to the α2β2 dimer. Heterotropic effectors like IHP may change the oxygen affinity of Hb through modulating the relative motion of the two dimers and then further altering the structure of heme binding regions.

Figure 1

Comparison of the symmetric axis orientations (a-d) and the switch region in α1β2 interface (e) of HbCO A in the T, R, R2 and solution conformations. (a). Distribution of the C₂ axes of different structures in a 3D frame. The angles shown in b, c and d are not drawn to scale and are enlarged for better visualization. The C₂ axes of the T, R, R2 and 20 solution conformations are in blue, red, magenta and black lines. The angles between the C₂ axes of the T structure and the 20 lowest energy NMR structures (b), between the C₂ axes of the R structure and the 20 lowest energy NMR structures (c), and between the C₂ axes of the R2 structure and the 20 lowest energy NMR structures (d). The average direction of the C₂ axes of the solution structures is denoted by a green arrow in b, c and d. The switch region in the T, R, R2 and one representative solution conformation are shown in blue, red, magenta and yellow respectively in (e). The backbone atoms of residues 38-44 in the α1 subunit are superimposed in order to illustrate the relative orientation of β2His97.

Figure 2

Chemical shift perturbations of backbone amides and side chain methyl groups by the binding of IHP to HbCO A. The chemical shift perturbations were defined as Δδ_av=[(Δδ_HN²+ Δδ_N²/25)/2]^0.5 for amide NH and Δδ_av=[(Δδ_HC²+ Δδ_C²/4)/2]^0.5 for methyl groups, where δ_HN, δ_N , δ_HC and δ_C are the chemical shift differences of amide ¹H, amide ¹⁵N, methyl ¹H, and methyl ¹³C between the samples in the presence of 3 mM IHP and in the absence of IHP. The empty regions represent no information available for residues in those regions because the NH and CH HSQC peaks of those residues were invisible or unassigned. The residues containing no methyl groups are located in the empty regions too. For Leu and Val, the data for γ₁ and δ₁ methyl groups are shown in blue bars while the data for γ₂ and δ₂ methyl groups in red bars. Other methyl groups are displayed in red bars. The dash lines represent the mean values over all residues with perturbation data.

Figure 3

Heme and its proximal methyl-containing residues in the α-chain and β-chain. Methyl carbon atoms displaying significant and insignificant chemical shift perturbations are shown in red and green, respectively.

Figure 4

IHP binding sites in the β-cleft (a), α-cleft (b) and center cavity (c). IHP molecule is shown in blue stick.

Figure 5

Order parameters S²_axis, describing the degree of spatial restriction of the C₃ symmetric axis for methyl groups in the α-chain (a) and β-chain (c) in the absence of IHP and in the α-chain (b) and β-chain (d) in the presence of IHP. The uncertainties of S²_axis for most methyl groups were about 0.01 and the maximum uncertainty was about 0.02. The open and filled circles represent pro-R and pro-S methyl groups for Val and Leu, respectively. Ala, Leu, Met, Thr and Val methyls are shown in magenta, red, green, light blue and black.

Figure 6

Relaxation dispersion profiles of the methyl groups with intrinsic conformational exchange in the absence and presence of IHP. The data collected in the samples with and without IHP are presented by ‘o’ and ‘*’, respectively. The solid lines are fitting curves assuming the two-state exchange model. The identity of each residue is indicated inside each panel.