Linkage of Interactions in Sickle Hemoglobin Fiber Assembly

The AB and GH regions of the α-chain are located in spatial proximity and contain a cluster of intermolecular contact residues of the sickle hemoglobin (HbS) fiber. We have examined the role of dynamics of AB/GH region on HbS polymerization through simultaneous replacement of non-contact Ala 19 and Ala 21 of the AB corner with more flexible Gly or rigid α-aminoisobutyric acid (Aib) residues. The polymerization behavior of HbS with Aib substitutions was similar to the native HbS. In contrast, Gly substitutions inhibited HbS polymerization. Molecular dynamics simulation studies of α-chains indicated that coordinated motion of AB and GH region residues present in native (Ala) as well as in Aib mutant was disrupted in the Gly mutant. The inhibitory effect due to Gly substitutions was further explored in triple mutants that included mutation of an inter-doublestrand contact (αAsn 78 → His or Gln) at the EF corner. Although the inhibitory effect of Gly substitutions in the triple mutant was unaffected in the presence of αGln 78 , His at this site almost abrogated its inhibitory potential. The polymerization studies of point mutants (αGln 78 → His) indicated that the inhibitory effect due to Gly substitutions in the triple mutant was synergistically compensated for by the polymerization-enhancing activity of His 78 . Similar synergistic coupling, between αHis 78 and an intra-double-strand contact point (α16) mutation located in the AB region, was also observed. Thus, two conclusions are made: ( i ) Gly mutations at the AB corner inhibit HbS polymerization by perturbing the dynamics of the AB/GH region, and ( ii ) perturbations of AB region (through changes in dynamics of the AB/GH region or abolition of a specific fiber contact site) that influence HbS polymerization do so in concert with α78 site at the EF corner. The overall results provide insights about the interaction-linkage between distant regions of the HbS tetramer in fiber assembly.