Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase

Abstract

The pathogen-associated 16S rRNA methyltransferase NpmA catalyzes m 1 A1408 modification to block the action of structurally diverse aminoglycoside antibiotics. Here, we describe the development of a fluorescence polarization binding assay and its use, together with complementary functional assays, to dissect the mechanism of NpmA substrate recognition. These studies reveal that electrostatic interactions made by the NpmA ␤2/3 linker collectively are critical for docking of NpmA on a conserved 16S rRNA tertiary surface. In contrast, other NpmA regions (␤5/␤6 and ␤6/␤7 linkers) contain several residues critical for optimal positioning of A1408 but are largely dispensable for 30S binding. Our data support a model for NpmA action in which 30S binding and adoption of a catalytically competent state are distinct: docking on 16S rRNA via the ␤2/3 linker necessarily precedes functionally critical 30S substrate-driven conformational changes elsewhere in NpmA. This model is also consistent with catalysis being completely positional in nature, as the most significant effects on activity arise from changes that impact binding or stabilization of the flipped A1408 conformation. Our results provide a molecular framework for aminoglycoside resistance methyltransferase action that may serve as a functional paradigm for related enzymes and a starting point for development of inhibitors of these resistance determinants.KEYWORDS RNA binding proteins, aminoglycosides, antibiotic resistance, methyltransferase, rRNA modification, ribosomes A minoglycosides are potent antimicrobial agents used for clinical treatment of life-threatening infections of both Gram-positive and Gram-negative bacteria and are also used routinely for both veterinary and growth promotion applications in agricultural settings (1, 2). Most aminoglycosides bind 16S rRNA helix 44 (h44) to induce conformational changes in the universally conserved nucleotides A1492 and A1493 in the ribosome decoding center. As a result, the bacterial ribosome is rendered unable to accurately discern cognate mRNA-tRNA pairing, thus impairing translational fidelity (3-9). More recent evidence has also suggested an additional 23S rRNA binding site for some aminoglycosides which disrupts intersubunit bridge B2, impacting a ribosomal conformational change required during elongation (10).Both aminoglycoside-producing and human-pathogenic bacteria can achieve high levels of resistance to aminoglycosides by reducing drug permeability or increasing efflux from the cell, enzymatic chemical modification of the drug, or mutation or chemical modification of the aminoglycoside binding site (4,11,12). In particular, S-adenosyl-L-methionine (SAM)-dependent 16S rRNA methyltransferases are the predominant resistance mechanism found in aminoglycoside-producing bacteria and are an increasing clinical concern with their continued emergence in major human patho-

Functionally critical residues in the aminoglycoside resistance-associated methyltransferase RmtC play distinct roles in 30S substrate recognition

“…1C,E). This value is comparable to the 60 nM affinity previously measured for the m 1 A1408 methyltransferase NpmA (13). Binding measurements were also performed with RmtA, RmtB, RmtD and RmtD2, which together with RmtC, represent each of the three subclades in the m 7 G1405 methyltransferase phylogenetic tree ( Fig.…”

“…m 7 G1405 methyltransferases bind 30S with similar affinity and at a site overlapping that of the m 1 A1408 methyltransferases-We previously developed a competition fluorescence polarization (FP) assay to measure the binding affinity of wild-type and variant NpmA proteins to define this methyltransferase's mechanism of 30S substrate recognition and m 1 A1408 modification (13). We speculated that the close proximity of nucleotides A1408 and G1405 in h44 ( Fig.…”

“…Both free and 30S-bound m 1 A1408 methyltransferases, including NpmA, have been extensively characterized, revealing the molecular basis of their specific substrate recognition and modification mechanisms (10)(11)(12)(13)(14)(15). These enzymes exploit a conserved 16S rRNA tertiary surface adjacent to helix 44 (h44) to dock on the 30S, explaining the requirement for intact 30S as their substrate.…”

“…These enzymes exploit a conserved 16S rRNA tertiary surface adjacent to helix 44 (h44) to dock on the 30S, explaining the requirement for intact 30S as their substrate. Two extended regions that connect the fifth/ sixth and sixth/ seventh β-strands of the methyltransferase core fold (β5/6 and β6/7 linkers, respectively) position key residues for recognition and stabilization of A1408 in a flipped conformation for methylation (10,13).…”

“…Here, we have extended the use of a 30S binding assay previously developed in our lab for studies of NpmA (13) to the m 7 G1405 methyltransferases. From these direct 30S binding measurements and a structure-guided mutagenesis strategy based on a new structure of a m 7 G1405 methyltransferase family member (RmtC), we develop a new model for 30S substrate recognition by the m 7 G1405 methyltransferases.…”