Mechanism of Ribosomal Subunit Recognition and Modification by the Mycobacterium tuberculosis Ribosomal RNA Methyltransferase TlyA Pubblico
Laughlin, Zane (Spring 2022)
Abstract
Antibiotics are a vital component of medicine today and have been extensively used since their discovery at the beginning of the 20th century. Since then, however, bacteria have developed resistance mechanisms to counter all classes of antibiotics. In 2019, it was estimated that antibiotic-resistant bacteria and fungi cause almost 3 million infections and 35,900 deaths per year in the United States. One mechanism of resistance to ribosome targeting antibiotics is the expression of ribosomal RNA (rRNA) methyltransferases that chemically modify the antibiotic binding site to reduce its binding. However, in the case of intrinsically-expressed ribosomal methyltransferase TlyA of Mycobacterium tuberculosis, modification of the ribosome by this enzyme instead confers susceptibility to tuberactinomycin antibiotics like capreomycin. TlyA incorporates 2’-O methylations on two bacterial ribosome nucleotides: 16S rRNA C1409 of the 30S subunit and 23S rRNA C1920 of the 50S subunit (E. coli numbering). The full-length structure of TlyA and how it recognizes and modifies these two sites in either of their different contexts, until now, was unknown.
In this work, we present the cryo-electron microscopy (cryo-EM) structure of a mycobacterial 50S subunit-TlyA complex trapped in a post-catalytic state using a S- adenosyl-L-methionine analog. Complementary functional analyses reveal a conserved surface of residues that spans both TlyA domains which recognizes the 23S rRNA. Specifically, conserved TlyA residues make critical contacts to unique tertiary structure at the base of 23S rRNA Helix 69 as well as surrounding the site of modification. The mechanism of modification determined from these studies positions the TlyA active site over the target nucleotide C1920 whose base is flipped from Helix 69. This work suggests that base flipping may be a common mechanism among rRNA methyltransferase enzymes even for those enzymes like TlyA which do not modify the nucleotide base. Finally, additional functional analyses on the 30S subunit suggest that the same 23S rRNA recognition surface of TlyA engages this second substrate, but with different dependencies on specific critical residues for binding.
Collectively, the work presented here details the mechanism by which TlyA recognizes and modifies its ribosomal substrates, particularly the 50S ribosomal subunit, through the use of structural and activity studies and contributes to the overall knowledge of the molecular mechanism of recognition and modification of other ribosomal methyltransferases.
Table of Contents
Table of Contents
Chapter 1: Introduction ............................................................................... 1
Antibiotics: Discovery and Usage ........................................................................2
Antibiotic Mechanism of Action .......................................................................... 3
Antibiotic Resistance Mechanisms......................................................................8
Drug Development and Strategies to Combat Resistance .................................. 13
Mycobacterium tuberculosis............................................................................. 14
Treatment of Mtb infection and drug resistance ............................................... 15
Ribose 2’-OH Ribosomal Methyltransferase TlyA.............................................. 16
Goals of this Research.......................................................................................20
References ........................................................................................................ 23
Chapter 2: Tuberactinomycin antibiotics: Biosynthesis, anti- mycobacterial action and resistance ........................................................27
Abstract ............................................................................................................29
Introduction .....................................................................................................29
Discovery and chemical structure of the tuberactinomycin antibiotics ............. 31
Elucidation of tuberactinomycin biosynthesis ..................................................32
Viomycin and capreomycin mechanism of antibiotic action.............................. 33
Clinical Use ....................................................................................................... 35
Mechanisms of Resistance to Tuberactinomycins .............................................36
Conclusions and Future Perspective ................................................................. 37
Acknowledgements ........................................................................................... 39
References ........................................................................................................ 39
Chapter 3: 50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA.....43
Abstract ............................................................................................................45
Signficance Statement.......................................................................................45
Introduction .....................................................................................................46
Results .............................................................................................................. 47
Determination of the 50S-TlyA complex structure .............................................................................. 47
TlyA NTD residues Arg6 and Arg20 exploit a complex rRNA structure for specific substrate recognition............................................................................................................................................. 49
TlyA CTD interactions with H69 position the methyltransferase domain for C2144 modification.... 50
TlyA employs a base flipping mechanism to position C2144 for ribose methylation...........................51
Insights into 30S subunit recognition and impact of TlyA clinical mutations .................................... 52
Discussion ........................................................................................................53
Materials and Methods .....................................................................................56
TlyA protein expression, purification and site-directed mutagenesis ................................................. 56
Isolation of Msm 50S and 30S subunits ............................................................................................... 57
Cryo-EM sample preparation, data collection and structure determination ...................................... 58
RT analysis of 23S rRNA methylation .................................................................................................. 59
Methyltransferase activity assays using [3H]-SAM .............................................................................. 59
Phylogenetic analysis of the TlyA protein family and residue conservation .......................................60
Acknowledgements ...............................................................................................................................60
References .............................................................................................................................................60
Supplementary Information ..................................................................................................................71
Chapter 4: TlyA structure, dynamics, and 30S subunit binding .............. 82
Introduction .....................................................................................................83
Mechanism of TlyA interaction with 30S subunit..............................................83
Towards a structure of apo-TlyA .......................................................................84
Determining the effect of SAM binding on the conformation and dynamics of TlyA. .................................................................................................................85
Conclusions ...................................................................................................... 87
References ........................................................................................................88
Chapter 5: Discussion............................................................................... 89
Structure of the 50S-TlyA complex and mechanism of modification ................. 91
Defining the complete 30S-binding surface of TlyA...........................................92
Determining the effect of SAM-binding on the conformation and function of TlyA. .................................................................................................................94
Base-flipping: a conserved mechanism in rRNA methyltransferases. ...............96
Impact of TlyA’s modifications on capreomycin binding. .................................. 97
Role of TlyA modifications in normal cell function. ..........................................98
Final remarks ..................................................................................................101
References ...................................................................................................... 103
Chapter 1
Table of Figures
Figure 1. Antibiotics target diverse processes and locations within the bacterial cell. .....................................................................................................................4
Figure 2. Ribosome-targeting antibiotics can inhibit each step of translation. ....6
Figure 3. Bacteria employ diverse mechanisms of antibiotic resistance. .............9
Figure 4. Modification sites of selected ribosomal RNA methyltransferases. .... 12
Figure 5. Knowledge of the TlyA modification sites and structure prior to this research. ........................................................................................................... 18
Figure 6. Difference in domain lengths between TlyA types I and II and FtsJ....20
Chapter 2
Figure 1. Ribosome targeting antibiotics bind at a variety of sites to inhibit translation. .......................................................................................................30
Figure 2. Chemical structures of the tuberactinomycin antibiotics........................32
Figure 3. Capreomycin and viomycin bind the 70S ribosome at subunit interface ribosome. .......................................................................................................... 34
Figure 4. 2’-O methyltransferase TlyA...............................................................38
Chapter 3
Figure 1. Cryo-EM map at 3.05 Å resolution of the 50S-TlyA complex. .............. 65
Figure 2. TlyA binds to 23S rRNA H69 and the adjacent rRNA junction via a surface of positively charged residues...............................................................66
Figure 3. The TlyA NTD recognizes a complex rRNA structure at the base of H69. .......................................................................................................................... 67
Figure 4. The TlyA CTD interacts with H69 surrounding the modification site..68
Figure 5. TlyA uses a base flipping mechanism to position C2144 for 2’-OH modification. ....................................................................................................69
Figure 6. TlyA has distinct residue dependencies for 30S methylation and is inactivated by clinically-identified resistance mutations...................................70
Fig. S1. Stabilization of the 50S-TlyA complex in a post-catalytic state using a SAM analog (NM6)............................................................................................ 72
Fig. S2. Workflow for cryo-EM structure determination. .................................. 73
Fig. S3. Cryo-EM map resolution analysis. ........................................................ 74
Fig. S4. Map corresponding to H54a differs among 3D classes. ......................... 75
Fig. S5. Superposition of TlyA with structural homologs. .................................. 76
Fig. S6. Interactions of TlyA NTD and CTD residues with 23S rRNA.................. 77
Fig. S7. Phylogenetic analysis of the TlyA enzyme family and residue conservation. .................................................................................................... 78
Fig. S8. Quality control of purified wild-type and variant TlyA proteins by thermal denaturation........................................................................................ 79
Fig. S9. Optimization of a [3H]-SAM methyltransferase assay for TlyA variants. ......................................................................................................................... 80
Fig. S10. Analysis of TlyA clinical variant proteins. ........................................... 81
Chapter 4
Figure. 1. TlyA-30S binding assay and analysis of TlyA variants. .......................84
Figure. 2. Crystallization of the free TlyA-SAM complex....................................85
Figure. 3. HDX-MS analysis of TlyA protein dynamics. .....................................86
Chapter 5
Figure 1. TlyA’s recognition surface varies between the 50S and 30S ribosomal subunits. ........................................................................................................... 93
Figure 2. Methyltransferase target sites on h44 of 30S ribosomal subunit. .......94
Figure 3. Potential structural consequences of C1920 methylation by TlyA on H69 conformation. ...........................................................................................99
Table of Tables
Chapter 3
Table 1. Table 1. Cryo-EM data collection, refinement and model validation for the 50S-TlyA complex. ......................................................................................64
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