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Aminoglycoside-Binding Site

The three-dimensional atomic structures of APH(3')-IIIa in complex with ADP and either the 4,6-disubstituted aminoglycoside kanamycin A or the 4,5-disubstituted aminoglycoside neomycin B have recently been determined (Fong and Berghuis 2002). Comparison of these structures with those of the apo, ADP and AMPPNP complexes of APH(3')-IIIa shows that most of the enzyme is rather rigid, with no evidence of gross domain movements associated with aminoglycoside binding. The most significant conforma-tional change observed is a shift in the C-terminal insertion known as the aminoglycoside-binding loop. The loop folds over towards the antibiotic

Fig. 4 An illustration of the APH(3')-IIIa aminoglycoside-binding pocket showing the con-formational changes that occur upon aminoglycoside binding. The backbone of the kana-mycin ternary structure of APH(3')-IIIa is shown as ribbon and the nucleotide cofactor and magnesium ions are drawn in balls-and-sticks, both in light grey. Kanamycin is shown as balls-and-sticks in grey. The antibiotic binding loop of the APH(3')-IIIa-ADP complex without bound aminoglycoside has been overlaid and coloured black. The movement of this loop is the largest conformational difference between the ternary and binary complexes

Fig. 4 An illustration of the APH(3')-IIIa aminoglycoside-binding pocket showing the con-formational changes that occur upon aminoglycoside binding. The backbone of the kana-mycin ternary structure of APH(3')-IIIa is shown as ribbon and the nucleotide cofactor and magnesium ions are drawn in balls-and-sticks, both in light grey. Kanamycin is shown as balls-and-sticks in grey. The antibiotic binding loop of the APH(3')-IIIa-ADP complex without bound aminoglycoside has been overlaid and coloured black. The movement of this loop is the largest conformational difference between the ternary and binary complexes substrate, interacts with the aminoglycoside and completes the binding pocket (Fig. 4). The majority of the residues that line the aminoglycoside-binding pocket are acidic, and consequently the pocket is negatively charged. Thus, the binding pocket complements the aminoglycoside substrates, which are invariably positively charged.

The aminoglycoside-binding site of APH(3')-IIIa can be considered to be composed of three distinct binding subsites. The first of these, subsite A, interacts with the central 2-deoxystreptamine ring of the aminoglycoside and the hexose substituted at the 4-position of this central ring (the prime ring). The second subsite (subsite B) interacts with groups substituted at the 6-po-sition of the 2-deoxystreptamine ring (the double prime ring). Consequently, this subsite is involved in binding only those substrates that have this type of modification, i.e. 4,6-disubstituted aminoglycosides. In contrast, the third subsite (subsite C) interacts with groups substituted at the 5-position of the central ring of 4,5-disubstituted aminoglycoside substrates.

Most of the hydrogen bond interactions observed between the aminoglycoside substrate and the APH(3')-IIIa enzyme are via subsite A. The central 2-deoxystrpetamine and prime rings that interact here are common to most aminoglycoside substrates and have been found to be the minimum essential components for antibacterial activity. There are fewer interactions between the aminoglycoside and subsites B and C. There is more variability in the parts of the aminoglycosides that interact here, and only functional groups that are conserved interact with the enzyme. While both subsites B and C are involved in fewer interactions than is subsite A, they differ significantly in size. This may be the consequence of the fact that the aminoglyco-side substituents at the 6-position (with which subsite B interacts) are single hexose rings, whereas substitutions at the 5-position (with which subsite C interacts) can be one, two or three sugar rings.

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