Restoring Allosterism with Compensatory Mutations in Hemoglobin

Abnormal human hemoglobins (Hbs) with amino acid substitutions in the α1β2interface have very high oxygen affinity and greatly reduce cooperativity in O2binding compared to normal human Hb. In such abnormal Hbs with mutations at position β99, the intersubunit hydrogen bonds between Asp-β99 and Tyr-α42 and between Asp- β99 and Asn-α97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occuring mutant Hb, Hb Kempsey (Asp- β99 → Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2binding properties of this recombinant double mutant Hb, Hb(Asp-β99 → Asn and Tyr-α42 → Asp) and have used1NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the α1β2subunit interface. Our results clearly show that the Tyr-α42 → Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-β99 → Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal Hbs associated with hemoglobinopathies.