Ligand binding studies in R67 dihydrofolate reductase and the evaluation of the anion-quadrupole interaction at a quantum mechanical level

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry related binding sites must exist for each of its ligands, various studies indicate only two molecules bind. To explore the role of various ligand substituents during binding, numerous analogs, inhibitors and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and Ki studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pKa, such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the para-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogs, the nicotinamide carboxamide is quite important.; Differences between binary and ternary complex formation are observed, indicating interligand pairing preferences. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.; The study of cation-pi interactions raises the question as to whether an edgewise pairing occurs with the aromatic rings of Phe, Tyr and Trp residues and the carboxylate groups of Glu and Asp. This pairing preference is termed an anion-quadrupole interaction. Quantum mechanical studies were done to evaluate the interaction energy and to deconvolute the energy terms involved. The model for this was the evaluation of benzene-formate pairs and as a reference, benzene with point charge(s). The results of the calculations show an electrostatic energy term with a distance dependency of 1/r3 for the anion-quadrupole effect. This is the predominant and favorable interaction energy term that is angle dependent. A contribution from the induction effect is also observed to potentiate this interaction.