Analysis of the π-π stacking interactions between the aminoglycoside antibiotic kinase APH(3′)-IIIa and its nucleotide ligands

A key contact in the active site of an aminoglycoside phosphotransferase enzyme (APH(3′)-IIIa) is a π-π stacking interaction between Tyr42 and the adenine ring of bound nucleotides. We investigated the prevalence of similar Tyr-adenine contacts and found that many different protein systems employ Tyr residues in the recognition of the adenine ring. The geometry of these stacking interactions suggests that electrostatics play a role in the attraction between these aromatic systems. Kinetic and calorimetric experiments on wild-type and mutant forms of APH(3′)-IIIa yielded further experimental evidence of the importance of electrostatics in the adenine binding region and suggested that the stacking interaction contributes ∼2 kcal/mol of binding energy. This type of information concerning the forces that govern nucleotide binding in APH(3′)-IIIa will facilitate inhibitor design strategies that target the nucleotide binding site of APH-type enzymes.