Effector-Induced Structural Fluctuation Regulates the Ligand Affinity of an Allosteric Protein: Binding of Inositol Hexaphosphate Has Distinct Dynamic Consequences for the T and R States of Hemoglobin

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Abstract
The present study reports distinct dynamic consequences for the T- and R-states of human normal adult hemoglobin (Hb A) due to the binding of a heterotropic allosteric effector, inositol hexaphosphate (IHP). A nuclear magnetic resonance (NMR) technique based on modified transverse relaxation optimized spectroscopy (TROSY) has been used to investigate the effect of conformational exchange of Hb A in both deoxy and CO forms, in the absence and presence of IHP, at 14.1 and 21.1 T, and at 37 °C. Our results show that the majority of the polypeptide backbone amino acid residues of deoxy- and carbonmonoxy-forms of Hb A in the absence of IHP is not mobile on the µs−ms time scale, with the exception of several amino acid residues, that is, β109Val and β132Lys in deoxy-Hb A, and α40Lys in HbCO A. The mobility of α40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between α40Lys and β146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of β109Val, which is located in the intradimer (α1β1 or α2β2) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify α40Lys in HbCO A, but does not significantly affect the flexibility of β109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (α1β2 or α2β1) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal β92His in the β-heme pocket, as well as some other residues located in the flexible joint (βC helix-αFG corner) and switch (αC helix-βFG corner) regions that play an important role in the dimer–dimer rotation of Hb during the oxygenation process. These findings suggest that, upon IHP binding, HbCO A undergoes a conformational fluctuation near the R-state but biased toward the T-state, apparently along the trajectory of its allosteric transition, accompanied by structural fluctuations in the heme pocket of the β-chain. In contrast, no significant perturbation of the dynamic features on the ms−µs time scale has been observed upon IHP binding to deoxy-Hb A. We propose that the allosteric effector-induced quaternary structural fluctuation may contribute to the reduced ligand affinity of ligated hemoglobin. Conformational exchange mapping of the β-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate (Rex) values, relative to those at lower fields, due to the enhanced effect of the local chemical shift anisotropy (CSA) fluctuation. A spring-on-scissors model is proposed to interpret the dynamic phenomena induced by the heterotropic effector, IHP.

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