Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacte rial enzyme that confers resistance to a range of aminoglycoside antibiotic s while exhibiting striking homology to eukaryotic protein kinases (ePK). T he structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 Angstro m resolution, respectively. Furthermore, refinement of the previously deter mined ADP complex was completed. The structure of the apoenzyme revealed al ternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite stru ctural similarity to protein kinases, there was no evidence of domain movem ent associated with nucleotide binding. This rigidity is due to the presenc e of more extensive interlobe interactions in the APH(3')-IIIa structure th an in the ePK's. Differences between the ADP and AMPPNP complexes are confi ned to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are d ifferences in metal coordination between the two nucleotide complexes. Comp arison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK 's suggests that the reaction mechanism of APH(3 ")-IIIa and related aminog lycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degree s. This alternate binding mode is likely a conserved feature among aminogly coside kinases and could be exploited for the structure-based drug design o f compounds to combat antibiotic resistance.