Inhibition of β(s)-chain dependent polymerization by synergistic complementation of contact site perturbations of α-chain: Application of semisynthetic chimeric α-chains

Mouse α_1-30-horse α_31-141 chimeric α-chain, a semisynthetic super-inhibitory α-chain, inhibits β(s)-chain dependent polymerization better than both parent α-chains. Although contact site sequence differences are absent in the α_1-30 region of the chimeric chain, the four sequence differences of the region α_17-22 could induce perturbations of the side chains at α₁₆, α₂₀ and α₂₃, the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse α_31-141 probably results in the super-inhibitory activity of the chimeric α-chain. The inhibitory contact site sequence differences, by themselves, could also exhibit similar synergistic complementation. Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin (LM) mutation [His20(α)→Gln], a contact site sequence difference, engineered into human-horse chimeric α-chain has been investigated to map such a synergistic complementation. Gln20(α) has little effect on the O₂ affinity of HbS, but in human-horse chimeric α-chain it reduces the O₂ affinity slightly. In the chimeric α-chain, Gln20(cl) increased sensitivity of the ββ cleft for the DPG influence, reflecting a cross-talk between the α₁β₁ interface and ββ cleft in this semisynthetic chimeric HbS. In the human α-chain frame, the polymerization inhibitory activity of Gln20(α) is higher compared with horse α_1-30, but lower than mouse α_1-30. Gln20(α) synergistically complements the inhibitory propensity of horse α_31-141. However, the inhibitory activity of LM-horse chimeric α-chain is still lower than that of mouse-horse chimeric α-chain. Therefore, perturbation of multiple contact sites in the α_1-30 region of the mouse-horse chimeric α-chain and its linkage with the inhibitory propensity of horse α_31-141 has been now invoked to explain the super-inhibitory activity of the chimeric α-chain. The 'linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into α-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.