How Allosteric Effectors Can Bind to the Same Protein Residue and Produce Opposite Shifts in the Allosteric Equilibrium

Abstract

Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.

Therapeutic Strategies to Alter the Oxygen Affinity of Sickle Hemoglobin

“…the effectors potency and/or direction of shift) is due not only to where the molecule binds, but also how it interacts with the Hb dimer-dimer interface to stabilize or destabilize that allosteric state. 48,49 …”

“…Interestingly, there are several aromatic aldehyde AEHs that also bind to the α-cleft to form Schiff-base with αVal1 nitrogen, but instead of left-shifting the OEC, they rather decrease Hb affinity for oxygen. 40,48,49,90 An ortho or para substituted carboxylate moiety (relative to the aldehyde functional group) in these right-shifters, e.g. , 5-formylsalicylic acid (5-FSA; Fig.…”

Design, Synthesis, and Biological Evaluation of Ester and Ether Derivatives of Antisickling Agent 5-HMF for the Treatment of Sickle Cell Disease

“…13,16,17 Unlike the above-mentioned aromatic aldehydes, there are other classes of aromatic aldehydes that bind Hb and preferentially stabilize the T-state relative to the R-state resulting in a decrease in Hb affinity for oxygen. 11,12,25,26 These compounds as expected promote sickling.…”

“…We have previously shown that aromatic aldehydes that form Schiff-base adduct with the N-terminal Val1 nitrogen and modify Hb may exhibit different allosteric behaviors (increase or decrease Hb affinity for oxygen), with the direction and magnitude of the allosteric shift dependent on preferential binding and/or stabilization of one Hb state (R or T) over the other. 11,25,26 For example, 5-HMF binds to both liganded and unliganded Hb, but significantly weaker to the latter; thus preferentially stabilizing the R-state and increasing Hb oxygen affinity. 11,15 It is likely that 5-NMFC and 5-CMFC bind and confer more stabilization to the T-state than 5-PMFC, in part explaining the significant differences in their P 50 and antisickling activities.…”

Identification of Novel Allosteric Regulators of Human‐Erythrocyte Pyruvate Kinase

“…These compounds were designed to bind at the FBP binding site, suggesting that an allosteric effector can bind to the same site, but produce opposite allosteric properties. We have reported this duality of action with binding at the same allosteric site in hemoglobin [51] [52]. Based on these results, a general hypothesis was proposed: that an effector ability to shift the allosteric equilibrium (i.e., the effectors potency) is due not only to where the molecule binds to, but also which residues it interacts with at the binding site This general concept can also be applied to R-PK, as we observed not only opposite effects, but also varying allosteric potencies among the tested compounds.…”