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Protein Engineering, Vol. 12, No. 12, 1105-1111, December 1999
© 1999 Oxford University Press

Inhibition of ßS-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Sonati Srinivasulu1, Ashok Malavalli1, Muthuchidambaran Prabhakaran3, Ronald L. Nagel1,2 and A.Seetharama Acharya1,2,4

1 Division of Hematology, Department of Medicine and 2 Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300, Morris Park Avenue, Bronx, NY 10461 and 3 Structural Bioinformatics, San Diego, CA 92127, USA

Mouse {alpha}1–30-horse {alpha}31–141 chimeric {alpha}-chain, a semisynthetic super-inhibitory {alpha}-chain, inhibits ßS-chain dependent polymerization better than both parent {alpha}-chains. Although contact site sequence differences are absent in the {alpha}1–30 region of the chimeric chain, the four sequence differences of the region {alpha}17-22 could induce perturbations of the side chains at {alpha}16, {alpha}20 and {alpha}23, the three contact sites of the region. A synergistic complementation of such contact site perturbation with that of horse {alpha}31–141 probably results in the super-inhibitory activity of the chimeric {alpha}-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({alpha})->Gln], a contact site sequence difference, engineered into human–horse chimeric {alpha}-chain has been investigated to map such a synergistic complementation. Gln20({alpha}) has little effect on the O2 affinity of HbS, but in human–horse chimeric {alpha}-chain it reduces the O2 affinity slightly. In the chimeric {alpha}-chain, Gln20({alpha}) increased sensitivity of the ßß cleft for the DPG influence, reflecting a cross-talk between the {alpha}1ß1 interface and ßß cleft in this semisynthetic chimeric HbS. In the human {alpha}-chain frame, the polymerization inhibitory activity of Gln20({alpha}) is higher compared with horse {alpha}1–30, but lower than mouse {alpha}1–30. Gln20({alpha}) synergistically complements the inhibitory propensity of horse {alpha}31–141. However, the inhibitory activity of LM–horse chimeric {alpha}-chain is still lower than that of mouse–horse chimeric {alpha}-chain. Therefore, perturbation of multiple contact sites in the {alpha}1–30 region of the mouse–horse chimeric {alpha}-chain and its linkage with the inhibitory propensity of horse {alpha}31–141 has been now invoked to explain the super-inhibitory activity of the chimeric {alpha}-chain. The `linkage-map' of contact sites can serve as a blueprint for designing synergistic complementation of multiple contact sites into {alpha}-chains as a strategy for generating super-inhibitory antisickling hemoglobins for gene therapy of sickle cell disease.

Keywords: chimeric HbS/HbS polymerization/intermolecular contact sites/linkage map/protein engineering/super-inhibitory {alpha}-chain

4 To whom correspondence should be addressed


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