Abstract
Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis. The clinically most prevalent bacterial resistance mechanism is their chemical modification by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). AadA from Salmonella enterica is an aminoglycoside (3″)(9) adenylyltransferase that O-adenylates position 3″ of streptomycin and position 9 of spectinomycin. We previously reported the apo-AadA structure with a closed active site. To clarify how AadA binds ATP and its two chemically distinct drug substrates, we here report crystal structures of WT AadA complexed with ATP, magnesium, and streptomycin and of an active-site mutant, E87Q, complexed with ATP and streptomycin or the closely related dihydrostreptomycin. These structures revealed that ATP binding induces a conformational change that positions the two domains for drug binding at the interdomain cleft and disclosed the interactions between both domains and the three rings of streptomycin. Spectinomycin docking followed by molecular dynamics simulations suggested that, despite the limited structural similarities with streptomycin, spectinomycin makes similar interactions around the modification site and, in agreement with mutational data, forms critical interactions with fewer residues. Using structure-guided sequence analyses of ANT(3″)(9) enzymes acting on both substrates and ANT(9) enzymes active only on spectinomycin, we identified sequence determinants for activity on each substrate. We experimentally confirmed that Trp-173 and Asp-178 are essential only for streptomycin resistance. Activity assays indicated that Glu-87 is the catalytic base in AadA and that the nonadenylating E87Q mutant can hydrolyze ATP in the presence of streptomycin.
Highlights
Streptomycin and spectinomycin are antibiotics that bind to the bacterial ribosome and perturb protein synthesis
Using isothermal titration calorimetry (ITC), we demonstrated that magnesium and ATP had to bind prior to aminoglycoside substrate, suggesting that ATP binding triggered a conforma
Using manual docking and molecular dynamics (MD) simulations, we present a model for how spectinomycin explores a partially different part of the active-site pocket
Summary
Cocrystallization of WT AadA with ATP and magnesium yielded well-diffracting crystals. The two molecules of AadA display close-toidentical structures (root mean square deviation (r.m.s.d.) of 0.1 Å over 261 C␣ atoms). This structure represents a native state of the enzyme prior to binding of aminoglycoside substrate. The interaction of Arg-192 with the bridging oxygen seems critical to obtain a magnesium-coordinating conformation of ATP because binding of nonhydrolyzable ATP analogues AMPCPP [12] and AMPNPP (data not shown) could not be detected. In the apo crystal structure, ATP- and magnesiumbinding residues of both domains were involved in interdomain interactions, explaining why that crystal form could not accommodate any ligand binding [12]. We can rationalize that some related structures, e.g. the structure of kanamycin nucleotidyltransferase in complex with AMPCPP [14], only display one magnesium ion because the bridging carbon of the ATP analogue affects the
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