Liganded Hb ζ₂β^S₂ Exhibits Antipolymer Activity Despite a T-like Quaternary Structure

Important therapeutic approaches to sickle-cell disease (SCD) are based upon the observation that the abnormal properties of Hb S (Hb α₂β^S₂) can be mitigated by exchanging the pathological β^S-globin subunit for a related β-like subunit. We previously demonstrated that exchange of the non-pathological α-globin subunit for a ζ-globin subunit (a developmentally silenced globin that can be derepressed both by natural and experimental conditions) inhibits deoxyHb S polymer assembly in vitro and reverses hematological, biochemical, and physiological characteristics of SCD in mouse models in vivo. While its therapeutic potential is clear, the underlying structural basis for the profound antipolymer activity of ζ-substituted Hb S (Hb ζ₂β^S₂) is less certain. X-ray crystallographic studies conducted at 1.95Å resolution revealed that liganded (CO-) Hb ζ₂β^S₂ is trapped in a tense (T-state) quaternary structure, rather than in a relaxed (R-state) structure that is characteristic of nearly all liganded hemoglobins. Specifically, CO-Hb ζ₂β^S₂ exhibited several intact T-state intersubunit salt-bridge/hydrogen-bond interactions, a preserved T-state ζ1-β2 (ζ2-β1) interface, and a characteristically enlarged T-state central water cavity and β-cleft. This structure wrongly predicts that liganded Hb ζ₂β^S₂ will be included, rather than excluded, from the deoxyHb S polymer; and suggests that changes in the positions or the biochemical identities of individual amino acids, rather than the overall quaternary structure of liganded Hb ζ₂β^S₂, are the chief determinants of its antipolymer activity.

To define key differences in the structures of T-state deoxyHb α₂β^S₂ and CO-Hb ζ₂β^S₂, we superposed their corresponding globin subunits and calculated the specific displacement of individual amino-acid residues as root mean square deviation (rmsd) values. Among β^S-chain residues, α→ζ exchange effects a significant 1.9Å shift in the position of the pathological β^SVal6, and correspondingly large displacements of βThr4 (2.2Å) and βAsn19 (1.4Å); each repositioning predicts weakening of an intermolecular interaction that would otherwise stabilize the deoxyHb S polymer. Similar superposition analyses of the α and ζ chains reveal a significant displacement of αPro114 (1.3Å), a well-described determinant of deoxyHb S polymerization that is conserved between the two α-like subunits. Three additional α-chain residues that stabilize the deoxyHb S polymer undergo nonconservative replacement in the ζ-globin chain, but are not materially repositioned: αHis20→ζGln (basic→neutral polar), αAsn68→ζAsp (neutral polar→acidic), and αAsn78→ζGly (neutral polar→neutral). While all three replacements are predicted to weaken or ablate intermolecular contacts, the αHis20→ζGln substitution is particularly noteworthy as it reproduces the specific mutation that defines the naturally occurring anti-sickling variant α^{Le Lamentin}.

Finally, we considered the possibility that ζ-substituted Hb S is fully excluded from the deoxyHb S polymer--and therefore reduces the rate of its assembly--by comparing the crystal packing of the two hemoglobins. While deoxyHb α₂β^S₂ packs in a familiar two-strand structure, CO-Hb ζ₂β^S₂ assembles into a unique trimeric arrangement comprising three lateral heterotetramers, each of which interacts with an axial heterotetramer that is constituent to a separate trimer assembly. This remarkable structure is sustained by intermolecular interactions that are distinct from those observed for deoxyHb S. Moreover, the calculated buried solvent-accessible surface area for CO-Hb ζ₂β^S₂ (4806Ų) is nearly two-fold higher than for deoxyHb α₂β^S₂ (2510Ų), suggesting that Hb ζ₂β^S₂ exists in solution as a stable trimer of heterotetramers, and validating the hypothesis that Hbs α₂β^S₂ and ζ₂β^S₂ do not co-assemble in solution.

In sum, our crystal analyses indicate that the antipolymer activities of liganded Hb ζ₂β^S₂ arise through movements in the positions of β^S-chain residues, and through changes in the identities of α-chain residues. Our studies also demonstrate a novel packing structure for T-state liganded Hb ζ₂β^S₂ that is consistent with its exclusion from the deoxyHb S polymer. These data account for the significant antipolymer activity of ζ-substituted Hb S, and recommend the utility of therapeutic approaches to SCD that are based upon α-globin subunit exchange.