Structural basis for the antipolymer activity of Hbζ2βs2trapped in a tense conformation

Abstract

The phenotypical severity of sickle-cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2βs2) to contain embryonic ζ-globin in place of adult α-globin subunits (Hb ζ2βs2). Crystallographical analyses of liganded Hb ζζ2βs2, 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 βs subunits of T-state deoxyHb α2βs2 and T-state CO-liganded Hb ζ2βs2 reveal significant displacements of both mutant βsVal6 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 ζ2βs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological βsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2βs2 suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ2βs2 appear to arise from both repositioning and replacement of specific α- and βs-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 ζ2βs2, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

Hemoglobin: Structure, Function and Allostery

“…It is clear that ligand binding to Hb ζ 2 β 2 s is effected mostly by tertiary structural changes within the larger T-or R state structures, providing insights into the contributions of tertiary and quaternary structures to cooperative Hb-O 2 ligand binding, as well as validating the hypothesis that Hb ligand affinity can be decoupled from overall quaternary structure (Henry et al 2002;Yonetani et al 2002;Yonetani and Tsuneshige 2003;Yonetani and Kanaori 2013). Moreover, the structure explains Hb ζ 2 β 2 s antipolymer activities by favoring an alternate T state structure that is excluded from pathological deoxygenated Hb S polymers (Safo et al 2015).…”

“…The structure displayed a central water cavity, dimer interface and salt-bridge/hydrogenbond interactions, β-cleft, etc. that are more typical for a tense conformation (Safo et al 2013(Safo et al , 2015. It is clear that ligand binding to Hb ζ 2 β 2 s is effected mostly by tertiary structural changes within the larger T-or R state structures, providing insights into the contributions of tertiary and quaternary structures to cooperative Hb-O 2 ligand binding, as well as validating the hypothesis that Hb ligand affinity can be decoupled from overall quaternary structure (Henry et al 2002;Yonetani et al 2002;Yonetani and Tsuneshige 2003;Yonetani and Kanaori 2013).…”

“…Several of the proposed allosteric models maintain the original MWC tenet that cooperative oxygen binding cannot occur in the absence of quaternary transition, consistent with the fact that a fully liganded T state hemoglobin heterotetramer, obtained from crystallizing ligated hemoglobin in solution, has never been reported. In a recent study, we reported the structure of such a uniquely fully liganded variant mammalian Hb ζ 2 β 2 s (formed from Hb S α 2 β 2 s by replacing adult α-globin with embryonic ζ-globin subunits), crystallized from CO-liganded Hb solution that remains trapped in a quaternary T state-like conformation (Safo et al 2013(Safo et al , 2015. Hb ζ 2 β 2 s inhibits polymerization of deoxygenated Hb S in vitro and ameliorates pathogenic features of sickle cell disease (SCD) in mouse models (He and Russell 2004a, b).…”

Rational Drug Design of Peptide-Based Therapies for Sickle Cell Disease

“…Targeting sickle hemoglobin for inhibitor design does not only aim to directly inhibit its aggregation into multi-stranded polymers, but also includes approaches that either result in the stabilization of the R conformation of the HbS molecule, or the destabilization of the T conformer [105,106]. Compounds whose antisickling properties are based on this concept include vanillin and pyridyl derivatives of vanillin, 5-hydroxymethylfurfural (5-HMF), and the recently approved voxelotor (GBT 440) [54,99,[106][107][108][109][110].…”