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Structural and functional studies of a toxin-antitoxin system involved in translational inhibition

Schureck, Marc (2016)
Dissertation (273 pages)
Committee Chair / Thesis Adviser: Dunham, Christine
Committee Members: Corbett, Anita ; Ortlund, Eric ; Rather, Phil N ; Reines, Daniel
Research Fields: Biochemistry; Microbiology; Molecular biology
Keywords: Toxin-antitoxin; Ribosome
Program: Laney Graduate School, Biological and Biomedical Sciences (Biochemistry, Cell & Developmental Biology)
Permanent url: http://pid.emory.edu/ark:/25593/rh3h5

Abstract

Bacteria regulate protein synthesis during environmental stress as a survival mechanism. One way translation is regulated is through cleavage of ribosome-bound mRNA by ribosome-dependent toxins. This mRNA cleavage stops the synthesis of the protein encoded by the cleaved-mRNA, conserves nutrients and likely plays an important, yet unknown, role in altering the spectrum of proteins translated during stress. A very unique feature of ribosome-dependent toxins is that they can recognize and cleave several mRNA sequences on the ribosome. In this dissertation, the molecular mechanism of recognition and cleavage of adenosine-rich mRNA codons by the Proteus vulgaris HigB toxin, which was originally identified on a drug-resistance plasmid from a P. vulgaris urinary tract infection, was investigated. Structural and biochemical studies reveal that the HigB toxin displays degenerate substrate specificity by creating two A-site nucleotide-binding pockets capable of interacting with numerous nucleotides. Surprisingly, the third nucleotide-binding pocket of HigB is adenosine-specific. Recognition of the third A-site nucleotide appears to be a distinct feature of ribosome-dependent toxins and likely influences which mRNAs are targeted for cleavage during environmental stress.

Ribosome-dependent toxins must be highly regulated. The HigA antitoxin binds to and inactivates the HigB toxin when cells are not in a stressed state. Structural investigation shows that HigA and HigB form a tetrameric complex consisting of two HigA proteins and two HigB proteins. This structure reveals that HigA does not inactivate HigB through direct interactions with the HigB active site, as observed in many other toxin-antitoxin complexes. Instead, HigA binding to HigB likely inhibits HigB by blocking association of HigB with the ribosome. The knowledge of how HigB activity is regulated and its unique specificity provides a molecular framework for scientists to uncover how ribosome-dependent toxins control translation during environmental stress.

Table of Contents

Chapter 1 ........................................................................................................................... 1

Introduction: Toxin-antitoxin systems............................................................................ 1

1.1 Abstract ................................................................................................................................ 1

1.2 Antibiotics............................................................................................................................. 2

1.3 Molecular mechanisms of antibiotic action ....................................................................... 3

1.4 Antibiotics and the ribosome .............................................................................................. 4

1.5 Bacteria that are antibiotic tolerant - Persisters............................................................... 6

1.6 Mechanisms of persister cell formation ............................................................................. 7

1.7 The bacterial stringent response ........................................................................................ 8

1.8 Activation of the stringent response through the lack of amino acids ............................ 8

1.9 Toxin-Antitoxin Systems ..................................................................................................... 9

1.10 Transcriptional regulation of toxin-antitoxin systems ................................................. 11

1.11 Molecular targets of toxin proteins ................................................................................ 13

1.12 Toxin-antitoxin systems and persister cell formation .................................................. 13

1.13 Other roles of toxin-antitoxin systems ........................................................................... 15

1.14 Translational inhibition during stress ........................................................................... 16

1.15 Ribosome-dependent toxins ............................................................................................ 16

1.16 The HigB-HigA toxin-antitoxin system ......................................................................... 18

1.17 Questions addressed ........................................................................................................ 19

1.18 References......................................................................................................................... 30

Chapter 2 ......................................................................................................................... 41

Structure of the P. vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex ................. 41

2.1 Abstract .............................................................................................................................. 42

2.2 Introduction ....................................................................................................................... 42

2.3 Experimental procedures .................................................................................................. 44

2.3a HigBA expression and purification ............................................................................... 45

2.3b Crystallization, X-ray data collection and structural determination of HigBA

complexes. ............................................................................................................................. 46

2.3c Size exclusion chromatography (SEC) assays .............................................................. 47

2.3d Electrophoretic mobility shift assay (EMSA) ............................................................... 48

2.3e Molecular modeling HigB on the 70S ribosome ........................................................... 48

2.4 Results ................................................................................................................................. 49

2.4a Structural determination of the HigB-(HigA)2-HigB complex. .................................... 49

2.4b HigB adopts a microbial RNase fold............................................................................. 50

2.4c The interface between HigA and HigB is novel............................................................ 51

2.4d HigA monomer contains an intact DNA binding domain. ............................................ 52

2.4e HigA mediates the formation of the HigB-(HigA)2-HigB complex.............................. 53

2.4f HigA does not mask the HigB active site. ..................................................................... 54

2.4g A HigB-(HigA)2-HigB tetramer is required to interact with its DNA operator. ........... 54

2.5 Discussion ........................................................................................................................... 56

2.6 Acknowledgments-............................................................................................................. 60

2.7 Footnotes............................................................................................................................. 60

2.8 References........................................................................................................................... 78

Chapter 3 ......................................................................................................................... 89

mRNA bound to the 30S subunit is a HigB endonuclease substrate .......................... 89

3.1 Abstract .............................................................................................................................. 90

3.2 Introduction ....................................................................................................................... 91

3.3 Results ................................................................................................................................. 93

3.3a HigB toxin can target the initiation step of translation.................................................. 93

3.3b Structural basis of HigB toxin recognition of the 30S subunit. .................................... 94

3.4 Discussion ........................................................................................................................... 99

3.5 Methods and materials .................................................................................................... 103

3.5a Strains and plasmids. ................................................................................................... 103

3.5b Purification of E. coli 30S ribosomes.......................................................................... 103

3.5c HigB expression and purification. ............................................................................... 104

3.5d mRNA cleavage assays. .............................................................................................. 105

3.5e Structural determination of the 30S-HigB complex. ................................................... 105

3.5f Bacterial growth assays................................................................................................ 106

3.6 Acknowledgements .......................................................................................................... 107

3.7 References......................................................................................................................... 116

Chapter 4 ....................................................................................................................... 123

Defining the mRNA recognition signature of a bacterial toxin protein................... 123

4.1 Abstract ............................................................................................................................ 125

4.2 Significance....................................................................................................................... 125

4.3 Introduction ..................................................................................................................... 126

4.4 Results & Discussion ....................................................................................................... 128

4.4a Structural determination of HigB-ribosome complexes. ............................................. 128

4.4b Recognition of the A site by HigB involves distortion of the mRNA. ....................... 129

4.4c A-site nucleotide requirements for HigB cleavage. .................................................... 131

4.4d Cross talk between A-site nucleotides drives efficient HigB recognition of mRNA.. 134

4.4e A single HigB residue modulates codon selectivity. ................................................... 135

4.5 Conclusions....................................................................................................................... 136

4.6 Materials & Methods....................................................................................................... 139

4.6a Strains and plasmids. ................................................................................................... 139

4.6b Sequence and structural alignments. ........................................................................... 139

4.6c Wild-type HigB, HigB Ξ”H92 and HigB N71A expression and purification. ............. 140

4.6d Structural determination of HigB. ............................................................................... 140

4.6e Structure Determination of 70S-HigB complexes....................................................... 141

4.6f mRNA cleavage assays. ............................................................................................... 142

4.6g Remodeling of the 70S-YoeB mRNA A-site mRNA. ................................................ 143

4.7 Acknowledgements .......................................................................................................... 143

4.8 References......................................................................................................................... 167

Chapter 5 ....................................................................................................................... 175

Mechanism of endonuclease cleavage by the HigB toxin .......................................... 175

5.1 Abstract ............................................................................................................................ 176

5.2 Introduction ..................................................................................................................... 176

5.3 Materials and methods .................................................................................................... 179

5.3a Strains and plasmids. ................................................................................................... 179

5.3b 70S purification, complex formation and structure determination of the 70S-HigB

precleavage state complex................................................................................................... 179

5.3c Bacterial growth or toxicity assays.............................................................................. 180

5.3d Single-turnover kinetic measurements. ....................................................................... 181

5.3e Structure determination of HigB variants.................................................................... 182

5.4 Results ............................................................................................................................... 183

5.4a Structure determination of the 70S - wild-type HigB complex................................... 183

5.4b Effect of HigB variants on growth suppression. ......................................................... 184

5.4c HigB residues His54, Asp90, Tyr91 and His92 are critical for mRNA cleavage. ...... 186

5.4d His92 is critical for optimal organization of the HigB active site. .............................. 187

5.5 Discussion ......................................................................................................................... 187

5.6 Acknowledgment ............................................................................................................. 190

5.7 Funding ............................................................................................................................. 190

Chapter 6 ....................................................................................................................... 215

Conclusion ..................................................................................................................... 215

6.1 Abstract ............................................................................................................................ 215

6.2 Introduction ..................................................................................................................... 216

6.3 Toxin activity can have varying effects on translation ................................................. 217

6.4 Potential ways in which toxins reshape the translational landscape .......................... 219

6.5 Towards an understanding of the roles of ribosome-dependent toxins ...................... 221

6.6 Molecular studies will aid the accurate annotation of ribosome-dependent toxins... 222

6.7 Mechanism of antitoxin inhibition of toxin function .................................................... 224

6.8 Molecular mechanisms of cleavage of mRNA by ribosome-dependent toxins .......... 230

6.9 Concluding remarks ........................................................................................................ 235

6.10 References....................................................................................................................... 249

Table of figures

Figure 1.1: Overview of the bacterial ribosome. .......................................................... 21

Figure 1.2: Overview of the translation process. ......................................................... 22

Figure 1.3: Schematic of difference between antibiotic-resistance and persistence. 24

Figure 1.4: Schematic of stringent response activation by uncharged tRNA entering

the ribosome. ................................................................................................................... 25

Figure 1.5: Schematic of toxin and antitoxin degradation. ......................................... 26

Figure 1.6: Antitoxins typically contain an N-terminal DNA-binding domain and a

C-terminal toxin-neutralization domain. ...................................................................... 27

Figure 1.7: Current model for conditional cooperativity............................................ 28

Figure 1.8: Translational inhibition by stoichiometric and enzymatic factors. ........ 29

Figure 2.1. X-ray crystal structure of the HigB-(HigA)2-HigB complex.................... 61

Figure 2.2. HigBA forms a heterotetramer in both crystal forms.............................. 63

Figure 2.3. Structural comparisons of HigA antitoxin. ............................................... 65

Figure 2.4. Highly conserved amino acids of HigB cluster in a concave, solvent

accessible surface. ........................................................................................................... 67

Figure 2.5. Minimal interface between HigB and HigA. ............................................. 69

Figure 2.6. P. vulgaris HigA-HigA interface is mediated mainly via hydrophobic

interactions and each monomer contains a HTH motif. ............................................. 70

Figure 2.7. The tetrameric HigB-(HigA)2-HigB complex containing two DNA

binding motifs is required for interactions with DNA................................................. 72

Figure 2.8. HigB-(HigA)2-HigB complex clashes with S12 and 16S rRNA in the A

site of the ribosome. ........................................................................................................ 74

Figure 2.9. HigB (green) alignment with RelE (white; PDB ID 3K1Q). .................... 75

Figure 3.1: HigB recognizes the 30S subunit.............................................................. 108

Figure 3.2: 30S crystal form likely prevents toxin-engaged mRNA in the A site. .. 110

Figure 3.3: Two basic patches on the surface of HigB mediate recognition of 16S

rRNA helices 18, 30 and 31. ......................................................................................... 111

Figure 3.4: Ribosome-dependent toxins recognize the head domain of the 30S

subunit............................................................................................................................ 113

Figure 3.5: Model for toxin recognition of the ribosome. ......................................... 114

Figure 4.1. Structural basis for HigB recognition of mRNA on the 70S ribosome. 145

Figure 4.2. Quality of 70S-HigB bound maps and models. ....................................... 147

Figure 4.3. Comparison of HigB in different states. .................................................. 149

Figure 4.4. Orientation of A-site decoding center nucleotides upon HigB binding to

the 70S. ........................................................................................................................... 151

Figure 4.5: HigB recognition of A-site mRNA nucleotides. ...................................... 153

Figure 4.6. Model for the +5 nucleotide recognition by HigB that predicts G5 and

U5 are incompatible. ..................................................................................................... 154

Figure 4.7. HigB demonstrates a clear preference at the third A-site nucleotide. .. 156

Figure 4.8. In vitro analyses of ribosome-dependent mRNA cleavage by HigB. ..... 157

Figure 4.9. Structural basis for toxin specificity at the +5 nucleotide position and

similarities to general RNases. ..................................................................................... 159

Figure 4.10. Rebuild of the 70S-YoeB structure. ....................................................... 161

Figure 4.11. A single conserved HigB residue drives sequence specificity at the +6

position. .......................................................................................................................... 163

Figure 5.1: Recognition of the ribosomal A site by endonuclease HigB. ................. 192

Figure 5.2: Difference electron density for the A-site mRNA and HigB. ................ 193

Figure 5.3. Identification of essential HigB residues. ................................................ 194

Figure 5.4: Analysis of HigB residues important for mRNA cleavage. ................... 196

Figure 5.5: Effect HigB variants on the rate of mRNA cleavage.............................. 197

Figure 5.6: HigB His92 is critical for preordering the HigB active site................... 198

Figure 5.7: Proposed mechanism of HigB-mediated mRNA degradation on the

ribosome......................................................................................................................... 199

Figure 5.8: Mechanistic differences in how ribosome-dependent toxins recognize the

A-site mRNA substrate. ................................................................................................ 200

Figure 6.1: Ribosome-dependent toxins have several potential roles in translational

regulation. ...................................................................................................................... 237

Figure 6.2: The HipB and HigA antitoxins may share a degradation tag. .............. 239

Figure 6.3: Toxins named HigB lack several key residues that define Proteus

vulgaris HigB. ................................................................................................................ 240

Figure 6.4: Antitoxin proteins interact with toxin proteins through diverse

mechanisms.................................................................................................................... 241

Figure 6.5: The RelEB toxin-antitoxin systems is transcriptionally regulated by

toxin to antitoxin ratios. ............................................................................................... 243

Figure 6.6: Working model for how transcriptional regulation by toxin to antitoxin

ratios limit active toxin to short periods. .................................................................... 244

Figure 6.7: Sequence conservation of ribosome-dependent toxins........................... 246

Figure 6.8: Highly conserved residues of individual toxin family members mainly

cluster around mRNA path.......................................................................................... 248

Table of tables

Table 2.1: Crystallographic data and refinement statistics…………………………76

Table 3.1: Crystallography statistics for the 30S-HigB structure…………………115

Table 4.1: 70S-HigB structures……………………………………………………....164

Table 4.2: HigB structure…………………………………………………………….165

Table 4.3: mRNAs used for structural and biochemical analysis………………….166

Table 5.1. DNA primers used…………………………………………………….......202

Table 5.2. 70S - HigB precleavage state structure…………………………………..205

Table 5.3: Summary of the effects of HigB mutations on the Kcat of mRNA

cleavage………………………………………………………………………………...206

Table 5.4: Crystallographic table of HigB variants…………………………………207

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