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

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 ·s-ms time scale, with the exception of several amino acid residues, that is, · 109Val and · 132Lys in deoxy-Hb A, and R40Lys in HbCO A. The mobility of R40Lys in HbCO A can be explained by the crystallographic data showing that the H-bond between R40Lys and · 146His in deoxy-Hb A is absent in HbCO A. However, the conformational exchange of ·109Val, which is located in the intradimer (R 1 · 1 or R 2 · 2 ) interface, is not consistent with the crystallographic observations that show rigid packing at this site. IHP binding appears to rigidify R40Lys in HbCO A, but does not significantly affect the flexibility of · 109Val in deoxy-Hb A. In the presence of IHP, several amino acid residues, especially those at the interdimer (R 1 · 2 or R 2 · 1 ) interface of HbCO A, exhibit significant conformational exchange. The affected residues include the proximal ·92His in the ·-heme pocket, as well as some other residues located in the flexible joint (·C helix-RFG corner) and switch (RC helix-·FG 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 ·-chain. In contrast, no significant perturbation of the dynamic features on the ms-·s 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 ·-chain of HbCO A observed at 21.1 T shows significantly increased scatter in the chemical exchange contribution to the transverse relaxation rate (R ex ) 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.

New Look at Hemoglobin Allostery

“…74,81,90,95 (ii) RDC measurements for HbCO A and a low oxygen-affinity mutant Hb, rHb (αV96W), in the absence and presence of IHP, have confirmed that the transition pathway 69-70 from the unliganded to the liganded state of hemoglobin is likely T → R → R2. (iii) Allosteric effectors, e.g., IHP, alter both the tertiary and quaternary structure of deoxy- and CO-Hb as well as the dynamics of the Hb molecule, thus affecting its ligand affinity.…”

“…The conformational exchange described here is consistent with the results from our backbone relaxation study, i.e., the IHP-induced conformational exchange has been detected in three regions, namely, the α 1 β 2 interface, heme pockets, and the presently putative IHP-binding sites. 90 Localized conformational exchange in the α 1 β 2 interface was also detected early on for the indole NH group of β37Trp in HbCO A in the presence of IHP. 95 …”

“…92 Conformational exchanges occur in several amino acid residues, 90 such as β109Val and β132Lys in deoxy-Hb A, and α40Lys in HbCO A.…”

“…Through the use of naturally occurring human mutant hemoglobins as well as of a large number of recombinant mutant Hbs produced by using our Hb expression plasmid, we have carried out an extensive array of structural, dynamic, kinetic, and functional studies. 67,81,86,90,96,120,131,138,153,156,168-176 Some of the observed structural, dynamic, kinetic and functional changes can be related to the mutations. Some of them can be small, but the mutation at one site could affect the conformation of distant as well as neighboring residues.…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…For example, the binding of BZF or IHP to CO-Hb complex decreases the CO association rate approximately four or eight times, respectively (Marden et al, 1990), and decreases the affinity of R state deoxy-Hb for oxygen . Coletta et al (1999a) have reported the α-and β-chain (Song et al, 2008).…”