Liganded Hb ζ2βS2 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 α 2 β S 2 ) 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 ζ 2 β S 2 ) is less certain. X-ray crystallographic studies conducted at 1.95A resolution revealed that liganded (CO-) Hb ζ 2 β S 2 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 ζ 2 β S 2 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 ζ 2 β S 2 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 ζ 2 β S 2 , are the chief determinants of its antipolymer activity. To define key differences in the structures of T-state deoxyHb α 2 β S 2 and CO-Hb ζ 2 β S 2 , 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 1.9A shift in the position of the pathological β S Val6, and correspondingly large displacements of βThr4 (2.2A) and βAsn19 (1.4A); 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 (1.3A), 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 α 2 β S 2 packs in a familiar two-strand structure, CO-Hb ζ 2 β S 2 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 ζ 2 β S 2 (4806A 2 ) is nearly two-fold higher than for deoxyHb α 2 β S 2 (2510A 2 ), suggesting that Hb ζ 2 β S 2 exists in solution as a stable trimer of heterotetramers, and validating the hypothesis that Hbs α 2 β S 2 and ζ 2 β S 2 do not co-assemble in solution. In sum, our crystal analyses indicate that the antipolymer activities of liganded Hb ζ 2 β S 2 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 ζ 2 β S 2 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. Disclosures Safo: Baxter and AesRx companies have licensed our patented antisickling compounds. Consulted with AesRx LLC during phase I clinical studies of the antisickling compound, 5HMF for the treatment of sickle cell disease: #7160910; #7119208 Patents & Royalties, Consultancy, Research Funding.