Insights into the solution structure of human deoxyhemoglobin in the absence and presence of an allosteric effector

Abstract

We present a nuclear magnetic resonance (NMR) study in solution of the structures of human normal hemoglobin (Hb A) in the deoxy or unligated form in the absence and presence of an allosteric effector, inositol hexaphosphate (IHP), using 15N-1H residual dipolar coupling (RDC) measurements. There are several published crystal structures for deoxyhemoglobin A (deoxy-Hb A), and it has been reported that the functional properties of Hb A in single crystals are different from those in solution. Carbonmonoxyhemoglobin A (HbCO A) can also be crystallized in several structures. Our recent RDC studies of HbCO A in the absence and presence of IHP have shown that the solution structure of this Hb molecule is distinctly different from its classical crystal structures (R and R2). To have a better understanding of the structure-function relationship of Hb A under physiological conditions, we need to evaluate its structures in both ligated and unligated states in solution. Here, the intrinsic paramagnetic property of deoxy-Hb A has been exploited for the measurement of RDCs using the magnetic-field dependence of the apparent one-bond 1H-15N J couplings. Our RDC analysis suggests that the quaternary and tertiary structures of deoxy-Hb A in solution differ from its recently determined high-resolution crystal structures. Upon binding of IHP, structural changes in deoxy-Hb A are also observed, and these changes are largely within the alpha1beta1 (or alpha2beta2) dimer itself. These new structural findings allow us to gain a deeper insight into the structure-function relationship of this interesting allosteric protein.

Figure 1

The correlation between the observed and calculated RDC values both in the presence and absence of IHP, based on the crystal structures 1A3N, 4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 1YHR, and 2DN2 for deoxy-Hb A. For details about these PDB files, see the Table 2. The data in the absence of IHP are shown in red and in the presence of IHP are shown in blue. Errors in the observed RDC are also shown and depicted in the same color as above.

Figure 2

The quality of fit results from the correlation plots is shown in Figure 1. The X-ray crystal structure file names are shown along the x-axis and their corresponding reduced χ² values in the absence (red) and presence (blue) of IHP are along the y-axis.

Figure 3

Summary of the fitting quality of our measured RDCs in the absence and presence of IHP to eight high-resolution x-ray crystal structures: 4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 1YHR, and 2DN2. X-ray structure file names are shown along the x-axis and the corresponding reduced χ² values of the fit to either the α₁β₁ dimer (in striped symbol) only or the whole tetramer (in filled symbol) are along the y-axis, red color for the structure in the absence of IHP and blue color for the structure in the presence of IHP.

Figure 4

Difference (absolute value) between the observed and the calculated RDCs when fitting to deoxy-Hb A using the PDB crystal structure 1XXT. The amino acid residue numbers for the α₁β₁ dimer (residues 1–287) followed by α₂β₂ dimer (residues 288–574) are shown along the x-axis and the corresponding absolute differences along the y-axis. RDCs of the sample containing IHP are shown in blue, whereas the IHP-free sample RDCs are in red.

Figure 5

600-MHz (¹H,¹⁵N) HSQC spectra of chain-specifically labeled-deoxy-Hb A with IHP (blue) and without IHP (red) at 35 °C: (A) (U-¹⁵N, ²H)-labeled α-chains and unlabeled β-chain-chains; and (B) unlabeled α-chains and (U-¹⁵N, ²H)-labeled β-chains. In all samples, the Hb concentration was ~1 mM in 50 mM sodium phosphate at pH 7.0 and the IHP samples also contained 5 mM IHP.