Structural basis for the antipolymer activity of Hb ζ₂β^s₂ trapped in a tense conformation

The phenotypical severity of sickle-cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α₂β^s₂) to contain embryonic ζ-globin in place of adult α-globin subunits (Hb ζ₂β^s₂). Crystallographical analyses of liganded Hb ζζ₂β^s₂, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α→ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the β^s subunits of T-state deoxyHb α₂β^s₂ and T-state CO-liganded Hb ζ₂β^s₂ reveal significant displacements of both mutant β^sVal6 and conserved β-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ₂β^s₂ additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological β^sVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ₂β^s₂ suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ₂β^s₂ appear to arise from both repositioning and replacement of specific α- and β^s-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ₂β^s₂, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.