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

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 (␣FeO 2 ) 2 -(␤Zn) 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.Hemoglobin (1) is a tetrameric protein, which plays a vital role in the transport of oxygen. It consists of two dimers of ␣ and ␤ subunits that reversibly bind and release oxygen (1). The description of this cooperative phenomenon has been most frequently 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. These distinct states are the fully unliganded T-state and the fully ligated R-state. Szabo and Karplus (3) modified the two-state model incorporating the stereochemical mechanism suggested by Perutz (4) for the T to R switch, and introduced ligation-induced tertiary changes within the T-state. In this extended model (MWC-SK), it was proposed that cooperativity still works through a ligation-induced shift in the equilibrium of states T and R, but the model attributed importance in the conformational switch to certain changes at the inter-and intrasubunit interfaces.

Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium

“…The shiftides act in a manner similar to the allosteric model (1), according to which ligand binding can shift an equilibrium toward R or T states, as in the case of hemoglobin (30-33): Heterotropic allosteric effectors lower the oxygen affinity of the T state upon binding to hemoglobin by binding to the T or R state and shifting the R/T equilibrium in favor of the bound state. Such effectors also affect the dimer-dimer affinity of hemoglobin and even lead to tetramer dissociation in extreme cases (34). The shiftides add an additional dimension to such allosteric inhibition because they modulate the equilibrium between various oligomeric states and not within a given oligomer.…”

Structures of haemoglobin from woolly mammoth in liganded and unliganded states

“…It has been demonstrated, for example, that Hb in the R state may have an affinity just as low as the T-state protein: the artificial effector L35 binds to both the T state and R state and reduces the oxygen affinity of both to the same level (Shibayama et al, 2002;Yokoyama et al, 2006). The textbook description of Hb lays strong emphasis on the allosteric switch of the protein, but it is now clear that dynamic effects are also at work, control-ling the oxygen affinity of the protein within each quaternary state (Schay et al, 2006;Ho et al, 2011;Yonetani et al, 2002).…”

SnO2–poly(diallyldimethylammonium chloride) films: Electrochemical evidence for heme protein absorption, denaturation, and demetallation

“…Hemoglobin consists of four subunits each of ca. 17 kD and dissociation into dimers or monomer subunits has been observed, for example, in the presence of high applied pressures or with suitable allosteric effectors [35]. It has been proposed recently that mesoporous films made from small 6 nm diameter TiO 2 nanoparticles inhibit the absorption of large hemoglobin proteins [36] and films made from larger ca.…”