Mechanisms of pathogen-associated 16S rRNA methyltransferase Npm Atarget recognition and enzymatic activity Open Access

Vinal, Kellie (2016)

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Antibiotic resistance remains a pervasive problem in the treatment of bacterial infections. The continual evolution of resistance mechanisms in bacteria treated with antibiotics, together with horizontal gene transfer between bacterial species, contribute significantly to this growing health concern. Ribosomal RNA (rRNA) methyltransferase enzymes of both drug-producer and pathogen origin are being reported with increased incidence and now threaten the clinical efficacy of many ribosome-targeting antibiotics. The 16S rRNA methyltransferase NpmA specifically methylates adenosine 1408 (A1408) in the aminoglycoside binding site of the bacterial ribosome small subunit (30S), conferring exceptionally high levels of resistance to structurally diverse aminoglycoside antibiotics. NpmA was the first m1A1408 methyltransferase enzyme isolated from a human pathogen and is capable of conferring an unprecedented level of resistance in diverse bacterial species. As such, there is an urgent need to understand NpmA's mechanisms of action as a platform for developing novel inhibitors of this emerging resistance determinant. The work presented here reveals the precise molecular mechanism employed by NpmA to recognize and modify its substrate, the 30S subunit. Through structural, functional, and biochemical analyses we define the molecular features necessary for NpmA to catalyze m1A1408 modification and ultimately confer resistance. Our crystal structure of NpmA bound to the 30S subunit, the first reported complex structure of its kind, captures NpmA in a "precatalytic" state in which the enzyme is poised for methyl transfer. We show that initial enzyme-substrate docking is driven by electrostatic interactions between the NpmA B2/3 linker and a conserved tertiary surface of the 30S subunit comprising three disparate 16S rRNA helices brought into proximity only upon 30S assembly. Docking of NpmA on the 30S subunit triggers precisely controlled structural reorganization of two other NpmA regions, the B5/6 and B6/7 linkers, which orient functionally critical residues to flip A1408 from helix 44 and stabilize this flipped conformation for methyl transfer. A newly developed fluorescence polarization binding assay, together with structural and biochemical assays, allowed us to specifically probe the NpmA-30S interaction and identify critical residues involved in substrate recognition and catalytic activity. These analyses revealed that catalysis by NpmA is mediated primarily by precise tertiary structure, particularly of the B6/7 linker, which plays crucial roles in base flipping of A1408 and catalysis. Taken together, our data provide a molecular framework for aminoglycoside-resistance rRNA methyltransferases that might serve as a functional paradigm for related enzymes and provide a starting point for inhibitor development to ultimately extend the efficacy of aminoglycosides in the clinic.

Table of Contents

CHAPTER 1: Introduction 1

Part I: The bacterial ribosome as an antibiotic target 2

Ribosome structure and assembly 2

Ribosome function 4

Antibiotics that interfere with ribosome function 8

Part II: Aminoglycosides 10

Aminoglycoside properties, action, and origin 10

Part III: Aminoglycoside resistance 14

Antibiotic resistance is ancient 14

Mechanisms and origins of aminoglycoside resistance 15

Part IV: Aminoglycoside-resistance 16S rRNA methyltransferases 15

Mechanism of resistance 15

Features of the A1408 vs. G1405 aminoglycoside-resistance 16S rRNA methyltransferase families 17

Part V: 16S rRNA methyltransferases identification and the rise of the 16S rRNA methyltransferase threat 18

Part VI: NpmA 19

Part VII: Clinical significance and epidemiology 23

Chapter 1 References 27

CHAPTER 2: Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA 35

Chapter 2 Introduction 37

Chapter 2 Results and Discussion 40

Directed RNA structure probing orients NpmA on h44 40

Overview of the 30S-NpmA-sinefungin complex 40

Identification of residues critical for NpmA activity 41

Sequence independent, tertiary contacts distant from the methylation site direct 30S-NpmA recognition 43

Pre-catalytic state shows A1408 flipped from h44 and poised for methylation 45

Mechanisms of molecular recognition, conformational adaptation and A1408 modification 46

Mechanistic conservation and variation among aminoglycoside-resistance 16S rRNA methyltransferases 48

Modification enzymes may target nucleotides in the decoding center by a common mechanism 49

Feasibility of horizontal gene transfer to other pathogens 51

Chapter 2 Materials and Methods 52

Chapter 2 Figures 54

Chapter 2 References 59

Chapter 2 Supplementary Information 64

CHAPTER 3: Substrate recognition and modification by a pathogen-associated aminoglycoside-resistance 16S rRNA methyltransferase 82

Chapter 3 Introduction 85

Chapter 3 Materials and Methods 87

Chapter 3 Results 91

A fluorescence assay to probe 30S-NpmA interaction 91

The NpmA β2/3 linker drives interaction with the 30S subunit 93

Contributions of the NpmA β5/6 and β6/7 linkers to 30S-NpmA binding affinity 96

Role of the β5/6 linker in 30S substrate recognition 97

Role of the β6/7 linker in SAM binding and 30S substrate recognition 98

Chapter 3 Discussion 100

Chapter 3 Tables and Figures 108

Chapter 3 References 116

Chapter 3 Supplementary Information 122

CHAPTER 4: Conclusion 128

Chapter 4 References 138

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