Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

Gusztáv Schay,László Smeller,Antonio Tsuneshige,Takashi Yonetani,Judit Fidy

doi:10.1074/jbc.m604216200

Abstract

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (alphaFeO2)2-(betaZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70-200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl-. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states.

Highlights

Quently derived from the Monod-Wyman-Changeux (MWC)2 two-state allosteric model [2] that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer
The well known Bohr effect and results reported for ClϪ, influencing the oxygen affinity of the T-state [14, 15], show that, in a broader sense, Hϩ and ClϪ can be considered as being members of the family of allosteric effectors
We show that the characteristic pressure of this transition, and the equilibrium constant (Kd0) of the reaction, is sensitive to the presence of allosteric effectors both in the R- and in the T-analogue state of HbA

Summary

Introduction

Quently derived from the Monod-Wyman-Changeux (MWC) two-state allosteric model [2] that attributes cooperativity to a rapid equilibrium between two conformations of distinct oxygen affinity of the whole tetramer. The modulation of the oxygen dissociation curves by allosteric effectors is addressed in the extended MWC model by the assumption that allosteric effectors bind to the somewhat larger central cavity of the T-state and stabilize this conformation This shifts the R/T equilibrium in favor of the T-state and lowers the overall affinity to oxygen [8, 13]. A docking and molecular dynamics simulation study in our laboratory [21] proposed models for the structure of HbA bound with the allosteric effectors DPG, IHP, and 2-{4-{(3,5-dichlorophenylcarbamoyl){methyl}-phenoxy}-2-methylpropionoc acid These results supported the model allowing for binding of effectors in the R-state and proposed primary binding sites in the central cavity of the R-state tetramer.

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