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. 2013 Mar 26;52(12):2108-17.
doi: 10.1021/bi301620x. Epub 2013 Mar 15.

Tertiary and quaternary allostery in tetrameric hemoglobin from Scapharca inaequivalvis

Luca Ronda  1 , Stefano Bettati, Eric R Henry, Tara Kashav, Jeffrey M Sanders, William E Royer, Andrea Mozzarelli
Affiliations

Tertiary and quaternary allostery in tetrameric hemoglobin from Scapharca inaequivalvis

Luca Ronda et al. Biochemistry. .

Abstract

The clam Scapharca inaequivalvis possesses two cooperative oxygen binding hemoglobins in its red cells: a homodimeric HbI and a heterotetrameric A2B2 HbII. Each AB dimeric half of HbII is assembled in a manner very similar to that of the well-studied HbI. This study presents crystal structures of HbII along with oxygen binding data both in the crystalline state and in wet nanoporous silica gels. Despite very similar ligand-linked structural transitions observed in HbI and HbII crystals, HbII in the crystal or encapsulated in silica gels apparently exhibits minimal cooperativity in oxygen binding, in contrast with the full cooperativity exhibited by HbI crystals. However, oxygen binding curves in the crystal indicate the presence of a significant functional inequivalence of A and B chains. When this inequivalence is taken into account, both crystal and R state gel functional data are consistent with the conservation of a tertiary contribution to cooperative oxygen binding, quantitatively similar to that measured for HbI, and are in keeping with the structural information. Furthermore, our results indicate that to fully express cooperative ligand binding, HbII requires quaternary transitions hampered by crystal lattice and gel encapsulation, revealing greater complexity in cooperative function than the direct communication across a dimeric interface observed in HbI.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Quaternary assembly of human and clam hemoglobins. Subunits are depicted as van der Waals spheres for main chain and heme atoms, with heme groups shown in red, E and F helices in cyan and the rest of the main chain in gray. (a) Human oxygenated HbA (PDB code 1HHO) is shown with α subunits in dark gray and β subunits in light gray. Note how the E and F helices are on the outside of the molecule. (b) Scapharca inaequivalvis tetrameric HbII-CO (PDB code 1SCT) is shown viewed approximately along its molecular dyad with A subunits in dark gray and B subunits in light gray. Note the extensive subunit pairings involving the E and F helices. (c) Scapharca inaequivalvis homodimeric HbI-CO (PDB code 3SDH) viewed approximately along its molecular dyad showing the extensive dimeric interactions involving the E and F helices.
Figure 2
Figure 2
Fo−Fc omit maps, contoured at 3 σ, for the heme region of two of the subunits in both unliganded and CO-liganded form. The top two figures show the unliganded heme region with expected structural features, based on earlier analyses of the homodimeric HbI. In all eight subunits, Phe F4 packs in the heme pocket contacting both heme and proximal histidine atoms characteristic of the low affinity “T” state. As well, density is clearly evident in the figure for two of the T-state specific interface water molecules. The lower two figures show the CO-liganded forms of one A and one B subunit. The density for Phe F4 in the CO ligated form for the chain A shown indicates the presence of two alternate conformations for this residue. In this subunit, and one other A-type subunit, both T-state and R-state conformations of Phe F4 are present. In the other two A-type subunits and all four B-type subunits, the side-chain of Phe F4 packs in the subunit interface as evident in the lower right figure and is characteristic of the high affinity R-state form of the homodimeric HbI.
Figure 3
Figure 3
Ligand-linked transitions of core interface water molecules in HbII. The images show portions of the E and F helices and heme groups along with spheres for key water molecules. Five water molecules that are unaltered by ligation are shown as dark blue, with the remaining water molecules as light blue/cyan. The arrangement of water molecules and side chains shown at the top for unliganded HbII is indistinguishable from the conformations observed in unliganded HbI. The water arrangement for HbII-CO is similar to that for HbI-CO, but not identical, showing additional water molecules and some variability among AB dimers. The water structure, along with the variation of Phe F4 in HbII-CO suggests that in these crystals HbII-CO does not obtain as pure an R-state structure as has been earlier observed with both oxygenated and CO-liganded HbI.
Figure 4
Figure 4
Alpha-carbon trace for HbII-CO (green) and unliganded HbII (purple) following alignment of one A chain (lower right). As can be seen, ligand binding results in rotations of all subunits, in a generally clockwise direction for the view shown. Subunit rotations illustrated here range from 3.8–6.5°.
Figure 5
Figure 5
Oxygen binding curve to HbII in a solution containing 100 mM phosphate, 1 mM EDTA, pH 7.0, 15 °C (open circles). Data points were fitted to the Hill equation (dashed line) with a p50 of 7.73 ± 0.12 torr and a Hill coefficient of 1.90 ± 0.05, and to the Adair equation (solid line) with dissociation constants: K1 = 0.0446 ± 0.0072 torr−1, K2 = 0.0502 ± 0.0268 torr−1, K3 = 0.1974 ± 0.1319 torr−1, K4 = 0.7524 ± 0.2348 torr−1.
Figure 6
Figure 6
Elution profiles of G-100 Sephadex size exclusion chromatography of solutions containing 100 μM (solid black line) and 2 μM HbA (dashed black line), and 107 μM (solid grey line) and 5.6 μM HbII (dashed grey line), carried out at 20 °C in 100 mM phosphate, 1 mM EDTA, pH 7.0.
Figure 7
Figure 7
Polarized absorption spectra of HbII crystals soaked in 62% Na+/K+ phosphate, pH 7, 15 °C, collected with the electric vector of the polarized incident light parallel to the c (b) and a (c) crystal axis for oxy- (solid lines), deoxy- (dashed lines) and met- (dotted lines) species. The calculated polarization ratio is reported in (a).
Figure 8
Figure 8
Hill plots derived from oxygen binding curves recorded along the c (a) and a (b) crystal axis for HbII crystals soaked in 62% Na+/K+ phosphate, pH 7, at 15 °C. The calculated p50s are 2.57 ± 0.18 and 1.57 ± 0.10 torr, respectively, and the Hill coefficients are 1.05 ± 0.07 and 1.14 ± 0.06, respectively.
Figure 9
Figure 9
Hill plots from oxygen binding curves to HbII in solution (open circles), or encapsulated in silica gel in T (open triangles up) and R state (closed squares). Fitting of the data points for T and R-state gels yields p50s of 25.6 ± 0.9 and 3.4 ± 0.1 torr with Hill coefficients of 0.85 ± 0.02 and 1.06 ± 0.02, respectively. For R state gels, fractional saturations at an oxygen pressure of 3.40 torr at increasing concentrations of phosphate from 100 to 400 and 600 mM are also reported (grey squares). Hill plots for HbII crystals oxygen binding curves measured along the c (dashed line) and a (dash-dot-dotted line) extinction directions are reported as grey lines (data from figure 7).
Figure 10
Figure 10
a. T-state gel data (closed circles) fitted to the Hill equation (dashed line) and assuming two equally populated binding sites with no cooperativity (n=1) (solid line). The two curves are indistinguishable. b. R-state gel data (open circles) fitted to the Hill equation (dashed line) and assuming two equally populated binding sites with n=1.44 and an A/B inequivalence of 4.9 (solid line).
Figure 10
Figure 10
a. T-state gel data (closed circles) fitted to the Hill equation (dashed line) and assuming two equally populated binding sites with no cooperativity (n=1) (solid line). The two curves are indistinguishable. b. R-state gel data (open circles) fitted to the Hill equation (dashed line) and assuming two equally populated binding sites with n=1.44 and an A/B inequivalence of 4.9 (solid line).
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