The first step in the structure determination of a protein using RDCs is the backbone resonance assignment. The polypeptide backbone resonance assignments of deoxy-Hb A, required for the interpretation of the backbone RDCs, were obtained using triple-resonance TROSY-based NMR experiments applied to chain-specifically labeled Hb samples (33
). Assignments of deoxy-Hb A was a challenging task for a high-molecular-weight paramagnetic protein even after the assignment of its ligated, diamagnetic counterpart (HbCO A) was already solved (39
). About 20% of the signals are shifted and/or severely broadened due to the presence of high-spin Fe+2
in the hemes of deoxy-Hb A. The presence of the hemes produces enhanced relaxation as well as causes Fermi and/or pseudo-contact interactions with nearby amino acids residues and the atoms on the porphyrins. The apparent 1DNH
were determined from the B02
dependence of the one-bond 15
H splittings (1JNH
), measured for deoxy-Hb A samples at four different magnetic fields (11.7, 14.1, 19.8, and 21.1 T). The field-induced RDCs are most apparent at the highest magnetic field used (900-MHz 1
H frequency or 21.1 T) as described previously (31
). summarizes the 1
N RDCs for the α1
dimer of deoxy-Hb A in the absence and presence of IHP in 50 mM sodium phosphate buffer at pH 7.0 and 35 °C. The reported RDCs are taken from measurements obtained at 21.1 T.
1H-15N RDC values for the α1β1 dimer of deoxy-Hb A in the absence and presence of IHP in 50 mM sodium phosphate buffer at pH 7.0 and 35 °C. The reported RDCs are taken from the measurements obtained at 21.1 T.
The peaks in HSQC and TROSY spectra of deoxy-Hb A appear to be better resolved than in the corresponding spectra of HbCO A. This is not simply the result of reduced peak widths, but is also the result of reduced crowding due to the absence of peaks in deoxy-Hb A spectra for residues whose amide NH groups are close (within ~11 Å) to the paramagnetic Fe atom of the heme. RDCs used to fit Eq. (1)
are restricted to those that are well resolved in the HSQC spectrum and correspond to amino acid residues located in the α-helical regions of the protein molecule. This selection, along with the missing peaks for groups close to the paramagnetic heme, results in a set of 69 RDCs for the αβ dimer, 28 in the α-chain and 41 in the β-chain. These are sufficient in number to allow evaluation of various structural models.
All published crystal structures of Hb A exhibit C2
symmetry. It is primarily the orientation of the α1
dimer relative to the α2
dimer, i.e., the orientation of the C
2 axis relative to α1
that varies between T and R states. The C
2 symmetry of deoxy-Hb A in solution is indicated by a single set of NMR backbone resonances for the two α- and the two β-chains, respectively. The α1
backbone coordinates of the well-defined helical structure segments superimpose relatively well between different unligated, T-state structures and ligated, R-state structures (R and R2) separately. The deoxy-Hb A structure does not superimpose well with the HbCO A structure (). The RMSD values between the four-selected X-ray structures of Hb A when the well-defined helical segments are superimposed are shown in a matrix form in . We have used two recent T-state structures for deoxy-Hb A and the R and R2 structures for HbCO A. This comparison can indicate that there are subtle differences at the tertiary structure level between deoxy-Hb A and HbCO A. In other words, the tertiary structures of T and R state Hb As are slightly different. The RMSD comparisons between a few selected crystal structures (two for deoxy-Hb A and two for HbCO A) are also shown in . As our preliminary results have shown (31
), the crystal structure of deoxy-Hb A, 1XXT, gives the best fit to our measured RDCs. However, the quality of the fit is still not as good as that obtained in our previous work on HbCO A. This is significant considering that HSQC and TROSY spectra with better quality and resolvability have been collected for deoxy-Hb A. For 1XXT, the α1
dimer itself produces a little better fit to the measured RDCs (RMSD=1.79 Hz) than does the full tetramer (RMSD=1.92 Hz) (see ). The small difference in the quality of the fit for the dimer and tetramer of deoxy-Hb A could be due to small quaternary structure changes in the solution structure of deoxy-Hb A. However, the overall lower quality than expected, implies that some of the disagreement between measured and calculated RDCs could arise from tertiary structure changes in the α- and/or β-chains of deoxy-Hb A in solution.
RMSD matrix between the structured regions in the α1β1 dimer of Hb A in Å units. The PDB IDs and the crystallization conditions for the T and R state structures are also given.
Fitting results obtained from the α1β1 dimer and the full tetramer of different deoxy-Hb A structures.
It has been reported that IHP binds to the central cavity of Hb A (5
) and that the central cavity in deoxy-Hb A is larger than in HbCO A (9
). Allosteric effectors are known to bind much tighter in deoxy- than in ligated-Hb (1
). Also, there are reports of multiple binding sites for IHP in HbCO A (40
). All of this could lead to more extensive tertiary structure changes in the deoxy-Hb. We describe our investigation of the structural differences observed in the presence and absence of IHP on deoxy-Hb A in following paragraphs.
Nine different X-ray crystal structures of deoxy-Hb A, with the resolution ranging from 1.25 to 2.6 Å, have been selected to compare and identify which crystal structure is closer to that in solution. Detailed information about these structure files is provided in , and the details of the RDC fit to these eight different structures are shown in . The correlation plots between the observed and calculated RDCs, both in the absence and presence of IHP, based on the crystal structures 1A3N, 4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 1YHR, and 2DN2 are shown in . The scatter in these plots is in all cases larger than the estimated experimental precision (shown by error bars in the figure), and the scatter is in general larger in the presence of IHP (blue symbols) than it is in the absence of IHP (red symbols). The fitting results are also summarized in , where a comparison of reduced chi-square (χ2) of the fit to the high-resolution X-ray crystal structures in absence and presence of IHP is presented. The reduced χ2 is defined as the χ2 divided by the number of degrees of freedom.
Various high-resolution crystal structures of deoxy-Hb A used in our analysis. The different names in this table are the standard PDB nomenclatures.
Figure 2 The quality of fit results from the correlation plots is shown in . 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 (more ...)
The data suggest that the solution structure of the T-state hemoglobin is slightly different from all known high-resolution crystal structures available so far. As we have mentioned earlier, out of all high-resolution deoxy-Hb A structures in the Protein Data Bank, 1XXT provides the best fit to our RDC data. This result is interesting, because the highest resolution structure (2DN2) at 1.25 Å reported by Tame’s group does not provide the best fit, emphasizing the fact that the structure in solution may be different.
The deviations of data from the crystal structures are even more significant in the presence of IHP. This suggests that there is a structure change in deoxy-Hb A upon IHP binding. This is not necessarily expected given the larger central cavity in the deoxy-Hb A. However, the crystal structure of deoxy-Hb A provided in 1XXT still gives the best match to experimental data.
Then, we pose the next question, of whether the structural changes induced by IHP are at the tertiary or quaternary structural levels or may be at both. We have fitted our measured RDCs to the α1β1 dimer as well as to the whole tetramer using eight out of the nine crystal structures (4HHB, 1HGA, 1KD2, 1RQ3, 1XXT, 1BZ0, 1YHR, and 2DN2, see for details). We have excluded the PDB file 1A3N because a more refined structure has been reported by the same research group (PDB: 2DN2). The values for the quality of the fit to our measured RDCs in the absence and presence of IHP with these eight high-resolution X-ray structures are summarized in and . The results for the α1β1 dimer, i.e., residues 1–287, are shown as continuous filled color, while the similar results for the entire tetramer, i.e. residues 1–574 are shown with same color with stripes. In both the dimer and the tetramer fitting results, the data for deoxy-Hb A with and without IHP are shown in blue and red colors, respectively. The differences between the reduced χ2 values between the dimer and tetramer fitting are very small whether we are using the data in the presence or absence of IHP. However, in either of the models (dimer or tetramer), the fit is better in the absence of IHP than in the presence of IHP. This is true regardless of the crystal structure used in the comparison. This is a clear indication of a significant change at the tertiary structure level. Analysis of the changes on an amino-acid-residue specific basis using just the crystal structure 1XXT is presented in . Here the absolute differences between the observed and the calculated RDCs when fit to Hb A using the PDB structure 1XXT for different residues both in presence and absence of IHP are shown. The RDCs of the sample containing IHP are shown in blue, whereas the IHP-free sample RDCs are in red. The 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. More residues in the β-chain undergo significant changes than in the β-chain indicating that more residues in the β1- and β2-chains undergo conformational changes upon IHP binding.
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 (more ...)
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 α (more ...)
Structure change evidenced by chemical shift perturbations
shows representative HSQC spectra at 14.1 T of the chain-specific labeled deoxy-Hb A samples used in this work. As one can see, the addition of IHP causes a number of cross peaks to shift, especially in the β-chain. The assignments of β-chain cross-peaks for deoxy-Hb A in presence of IHP are very straightforward (33
). The assignments of cross-peaks from the β-chain which undergo significant change in peak position in HSQC spectra were confirmed by the use of 3D 15
N-TROSY-NOESY spectra recorded at 35 °C and 60-ms mixing time. It appears that IHP causes shifts mostly of cross-peaks from the amino acid residues located in the β-chain of deoxy-Hb A. This is in contrast to our previous work on the effect of IHP on HbCO A (8
), where we observed chemical shift changes distributed across both α- and β-chains. However, the current findings of a the larger effect of IHP on the β-chain of deoxy-Hb A are consistent with the crystallographic results on the binding of 2,3-BPG to deoxy-Hb A reported by Arnone (4
). The differences between CO and deoxy behavior may result from the tendency of the smaller central cavity in ligated-Hb to more easily propagate changes throughout the protein molecule.
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-15 (more ...)
Implications of the present study for the structure-function relationship in hemoglobin
There are eight unique crystal structures of deoxy-Hb A that have been deposited in the Protein Data Bank with resolution ranging from 1.25 to 2.6 Å (see and and ). The crystal structure for 1A3N has recently been refined and its refined structure is known as 2DN2 (21
). There are several interesting questions regarding these crystal structures. Does each of them exist as a discrete structure in solution? If so, what is the relative proportion for each of these structures in solution? Does each one of them exhibit its specific functional properties? Do they interconvert among themselves and if so, what is the time scale for this process in solution? As described in this paper, the solution structure of deoxy-Hb A as measured by the RDC method appears to be different from the nine crystal structures, with the crystal structure of 1XXT giving the best fit to our RDC data (). Does this imply that something close to the 1XXT structure is the dominant structure for deoxy-Hb A in solution, or is the apparent structure a result of more uniform averaging of data over the representative crystal structures? With respect to the latter point, a single set of cross-peaks is observed for nearly all residues. Hence, either one structure must dominate, or exchange among structures must be fast on the NMR timescale.
We can test the viability of a model in which uniform sampling of the various X-ray structures exists by using the averaging capabilities of the program, REDCAT (42
dimers from seven of the unliganded X-ray structures (excluding the mutant 1YHR) were superimposed in a manner that led to overlap of the hemes in each structure, hydrogens were added to the amide sites using standard geometries, and the combined set of new coordinates was used to find the alignment parameters that best fit a model in which each of the seven structures was populated equally. The back-calculated RDCs from this model showed an RMSD relative to experiment of 1.88 Hz. This is only slightly worse than the RMSD of the 1XXT structure and it is significantly better than the average RMSD over the set of structures, 2.07. Of course, there is no reason to expect equal sampling of the X-ray structures and it is likely that populations could be adjusted to produce a better fit.
Recently, we have carried out a Model-free based NMR dynamics studies for the polypeptide backbone amide N-H bond vectors for both the deoxy- and carbonmonoxy forms of Hb A, using 15
N-relaxation parameters and heteronuclear nuclear Overhauser effects measured at 29 and 34 °C and at 11.7 and 14.1 Tesla (43
). We have found that in both deoxy- and carbonmonoxy froms of Hb A, the amide N-H bonds of most amino acid residues are rigid on the fast time scale (ns-ps), except for the loop regions and certain helix-helix connections. The C-terminal β146His has been postulated to play an important role in the allostery of Hb A. Based on X-ray crystallographic data as well as its ability to form salt bridges and H-bonds in the deoxy form (9
). Our backbone dynamics data indicate that this residue is rigid in the deoxy-Hb A, but it is free from restrictions to its backbone motions in the CO form, consistent with the X-ray crystallographic findings. We have found that in the deoxy form, α31Arg and β123Thr, neighbors in the intra-dimer (α1
) interface, exhibit stiffening upon CO binding. We have also found that there is considerable flexibility in the α1
interface, e.g., B, G, and H helices and the GH corner and that several amino acid residues (e.g., β109Val) at this interface appears to be involved in a conformational exchange process in the deoxy form. We have already reported that the solution structure of HbCO A is a dynamic ensemble of the R and R2 crystal structures (23
). While additional research is needed to gain a deeper insight, there is sufficient information to suggest that a dynamic ensemble may exist in the deoxy form as well.
In conclusion, the plasticity and dynamic picture of hemoglobin is consistent with the emerging view of allostery as a change in population distribution of an ensemble of structures, rather than the equilibrium of only two discrete conformations, upon binding of ligand (6
). Hence, in order to understand how hemoglobin functions as an efficient oxygen carrier in our physiological system, we need to know the structure, dynamics, and function of this protein in solution.