Abstract
The phenotypical severity of sickle-cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α(2)β(s)(2)) to contain embryonic ζ-globin in place of adult α-globin subunits (Hb ζ(2)β(s)(2)). Crystallographical analyses of liganded Hb ζζ(2)β(s)(2), 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 α(2)β(s)(2) and T-state CO-liganded Hb ζ(2)β(s)(2) reveal significant displacements of both mutant β(s)Val6 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 ζ(2)β(s)(2) additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological β(s)Val6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ(2)β(s)(2) suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ(2)β(s)(2) 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 ζ(2)β(s)(2), and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.