Interspecies hybrid HbS: complete neutralization of val6(β)-dependent polymerization of human β-chain by pig α-chains

Interspecies hybrid HbS (α2Pβ2S), has been assembled in vitro from pig α-globin and human βS-chain. The α2Pβ2S retains normal tetrameric structure (α2β2) of human Hb and an O2 affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O2 affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The 1H-NMR spectroscopy detected distinct differences between the heme environments and α1β1 interfaces of pig Hb and HbS, while their α1β2 interfaces appear very similar. The interspecies hybrid α2Hβ2P resembles pig Hb; the pig β-chain dictated the conformation of the heme environment of the human α-subunit, and to the α1β1 interfaces of the hybrid. In the α2Pβ2S hybrid, βS-chain dictated the conformation of human heme environment to the pig α-chain in the hybrid; but the conformation of α1β1 interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the α1β2 interface conformation is identical to that of HbS. More important, the α2Pβ2S does not polymerize when deoxygenated; pig α-chain completely neutralizes the βS-chain dependent polymerization. The polymerization inhibitory propensity of pig α-chain is higher when it is present in the cis αPβS dimer relative to that in a trans αPβA dimer. The semisynthetically generated chimeric pig-human and human-pig α-chains by exchanging the α1–30 segments of human and pig α-chains have established that the sequence differences of pig α31–141 segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the α-chain surfaces of HbS and α2Pβ2S suggests that the differences in electrostatic potential energy surfaces on the α-chain of α2Pβ2S relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig α-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric α-chains can serve as blueprints for the design of a new generation of variants of α-chain(s) suitable for the gene therapy of sickle cell disease.