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

Nosrati, Meisam; Dey, Debayan; Mehrani, Atousa; Strassler, Sarah E.; Zelinskaya, Natalia; Hoffer, Eric D.; Stagg, Scott M.; Dunham, Christine M.; Conn, Graeme L.

doi:10.1074/jbc.RA119.011181

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

Journal Article · Tue Oct 08 00:00:00 EDT 2019 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.RA119.011181· OSTI ID:1577157

Nosrati, Meisam ^[1]; ^[1]; Mehrani, Atousa ^[2]; Strassler, Sarah E. ^[1]; Zelinskaya, Natalia ^[1]; Hoffer, Eric D. ^[1]; ^[2]; ^[1]; ^[1]

Emory Univ., Atlanta, GA (United States)
Florida State Univ., Tallahassee, FL (United States)

Methylation of the small ribosome subunit rRNA in the ribosomal decoding center results in exceptionally high-level aminoglycoside resistance in bacteria. Enzymes that methylate 16S rRNA on N7 of nucleotide G1405 (m⁷G1405) have been identified in both aminoglycoside-producing and clinically drug-resistant pathogenic bacteria. Using a fluorescence polarization 30S-binding assay and a new crystal structure of the methyltransferase RmtC at 3.14 Å resolution, here we report a structure-guided functional study of 30S substrate recognition by the aminoglycoside resistance-associated 16S rRNA (m⁷G1405) methyltransferases. We found that the binding site for these enzymes in the 30S subunit directly overlaps with that of a second family of aminoglycoside resistance-associated 16S rRNA (m¹A1408) methyltransferases, suggesting that both groups of enzymes may exploit the same conserved rRNA tertiary surface for docking to the 30S. Within RmtC, we defined an N-terminal domain surface, comprising basic residues from both the N1 and N2 subdomains, that directly contributes to 30S-binding affinity. In contrast, additional residues lining a contiguous adjacent surface on the C-terminal domain were critical for 16S rRNA modification but did not directly contribute to the binding affinity. The results from our experiments define the critical features of m⁷G1405 methyltransferase–substrate recognition and distinguish at least two distinct, functionally critical contributions of the tested enzyme residues: 30S-binding affinity and stabilizing a binding-induced 16S rRNA conformation necessary for G1405 modification. Our study sets the scene for future high-resolution structural studies of the 30S-methyltransferase complex and for potential exploitation of unique aspects of substrate recognition in future therapeutic strategies.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC)

Contributing Organization:: Advanced Photon Source (APS), Argonne National Laboratory (ANL), Argonne, IL (US)

Grant/Contract Number:: W-31109-ENG-38

OSTI ID:: 1577157

Journal Information:: Journal of Biological Chemistry, Journal Name: Journal of Biological Chemistry Journal Issue: 46 Vol. 294; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

References (59)

Modeling and experimental analyses reveal a two-domain structure and amino acids important for the activity of aminoglycoside resistance methyltransferase Sgm Vlahoviček, Gordana Maravić; Čubrilo, Sonja; Tkaczuk, Karolina L. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1784, Issue 4 https://doi.org/10.1016/j.bbapap.2007.09.009	journal	April 2008
Expansion of the aminoglycoside-resistance 16S rRNA (m1A1408) methyltransferase family: Expression and functional characterization of four hypothetical enzymes of diverse bacterial origin Witek, Marta A.; Conn, Graeme L. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1844, Issue 9 https://doi.org/10.1016/j.bbapap.2014.06.012	journal	September 2014
A Unique RNA Fold in the RumA-RNA-Cofactor Ternary Complex Contributes to Substrate Selectivity and Enzymatic Function Lee, Tom T.; Agarwalla, Sanjay; Stroud, Robert M. Cell, Vol. 120, Issue 5 https://doi.org/10.1016/j.cell.2004.12.037	journal	March 2005
Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: An update Wachino, Jun-ichi; Arakawa, Yoshichika Drug Resistance Updates, Vol. 15, Issue 3 https://doi.org/10.1016/j.drup.2012.05.001	journal	June 2012
Aminoglycoside Resistance Doi, Yohei; Wachino, Jun-ichi; Arakawa, Yoshichika Infectious Disease Clinics of North America, Vol. 30, Issue 2 https://doi.org/10.1016/j.idc.2016.02.011	journal	June 2016
Aminoglycoside Resistance Wachino, Jun-Ichi; Doi, Yohei; Arakawa, Yoshichika Infectious Disease Clinics of North America, Vol. 34, Issue 4 https://doi.org/10.1016/j.idc.2020.06.002	journal	December 2020
Do we still need the aminoglycosides? Durante-Mangoni, Emanuele; Grammatikos, Alexandros; Utili, Riccardo International Journal of Antimicrobial Agents, Vol. 33, Issue 3 https://doi.org/10.1016/j.ijantimicag.2008.09.001	journal	March 2009
Prevalence and molecular characterisation of New Delhi metallo-β-lactamases NDM-1, NDM-5, NDM-6 and NDM-7 in multidrug-resistant Enterobacteriaceae from India Rahman, Mohibur; Shukla, Sanket Kumar; Prasad, Kashi Nath International Journal of Antimicrobial Agents, Vol. 44, Issue 1 https://doi.org/10.1016/j.ijantimicag.2014.03.003	journal	July 2014
Structural Bases for 16 S rRNA Methylation Catalyzed by ArmA and RmtB Methyltransferases Schmitt, Emmanuelle; Galimand, Marc; Panvert, Michel Journal of Molecular Biology, Vol. 388, Issue 3 https://doi.org/10.1016/j.jmb.2009.03.034	journal	May 2009
FindEM—a fast, efficient program for automatic selection of particles from electron micrographs Roseman, A. Journal of Structural Biology, Vol. 145, Issue 1-2 https://doi.org/10.1016/j.jsb.2003.11.007	journal	January 2004
Automated molecular microscopy: The new Leginon system Suloway, Christian; Pulokas, James; Fellmann, Denis Journal of Structural Biology, Vol. 151, Issue 1 https://doi.org/10.1016/j.jsb.2005.03.010	journal	July 2005
EMAN2: An extensible image processing suite for electron microscopy Tang, Guang; Peng, Liwei; Baldwin, Philip R. Journal of Structural Biology, Vol. 157, Issue 1 https://doi.org/10.1016/j.jsb.2006.05.009	journal	January 2007
Appion: An integrated, database-driven pipeline to facilitate EM image processing Lander, Gabriel C.; Stagg, Scott M.; Voss, Neil R. Journal of Structural Biology, Vol. 166, Issue 1 https://doi.org/10.1016/j.jsb.2009.01.002	journal	April 2009
DoG Picker and TiltPicker: Software tools to facilitate particle selection in single particle electron microscopy Voss, N. R.; Yoshioka, C. K.; Radermacher, M. Journal of Structural Biology, Vol. 166, Issue 2 https://doi.org/10.1016/j.jsb.2009.01.004	journal	May 2009
CTFFIND4: Fast and accurate defocus estimation from electron micrographs Rohou, Alexis; Grigorieff, Nikolaus Journal of Structural Biology, Vol. 192, Issue 2 https://doi.org/10.1016/j.jsb.2015.08.008	journal	November 2015
Effects of the 1- N -(4-Amino-2 S -hydroxybutyryl) and 6′- N -(2-Hydroxyethyl) Substituents on Ribosomal Selectivity, Cochleotoxicity, and Antibacterial Activity in the Sisomicin Class of Aminoglycoside Antibiotics Sonousi, Amr; Sarpe, Vikram A.; Brilkova, Margarita ACS Infectious Diseases, Vol. 4, Issue 7 https://doi.org/10.1021/acsinfecdis.8b00052	journal	April 2018
Aminoglycoside Antibiotics in the 21st Century Becker, Bernd; Cooper, Matthew A. ACS Chemical Biology, Vol. 8, Issue 1 https://doi.org/10.1021/cb3005116	journal	November 2012
Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens Matsushita, Takahiko; Sati, Girish C.; Kondasinghe, Nuwan Journal of the American Chemical Society, Vol. 141, Issue 12 https://doi.org/10.1021/jacs.9b01693	journal	February 2019
Destination of aminoglycoside antibiotics in the ‘post-antibiotic era’ Takahashi, Yoshiaki; Igarashi, Masayuki The Journal of Antibiotics, Vol. 71, Issue 1 https://doi.org/10.1038/ja.2017.117	journal	October 2017
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination Punjani, Ali; Rubinstein, John L.; Fleet, David J. Nature Methods, Vol. 14, Issue 3 https://doi.org/10.1038/nmeth.4169	journal	February 2017
MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul Nature Methods, Vol. 14, Issue 4 https://doi.org/10.1038/nmeth.4193	journal	February 2017
PARP1 exhibits enhanced association and catalytic efficiency with γH2A.X-nucleosome Sharma, Deepti; De Falco, Louis; Padavattan, Sivaraman Nature Communications, Vol. 10, Issue 1 https://doi.org/10.1038/s41467-019-13641-0	journal	December 2019
Cavin1 intrinsically disordered domains are essential for fuzzy electrostatic interactions and caveola formation Tillu, Vikas A.; Rae, James; Gao, Ya Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-21035-4	journal	February 2021
Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs Bepler, Tristan; Morin, Andrew; Rapp, Micah Nature Methods, Vol. 16, Issue 11 https://doi.org/10.1038/s41592-019-0575-8	journal	October 2019
The co-transfer of plasmid-borne colistin-resistant genes mcr-1 and mcr-3.5, the carbapenemase gene blaNDM-5 and the 16S methylase gene rmtB from Escherichia coli Long, Haiyan; Feng, Yu; Ma, Ke Scientific Reports, Vol. 9, Issue 1 https://doi.org/10.1038/s41598-018-37125-1	journal	January 2019
Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen Ou, Li; Kong, Wing-Pui; Chuang, Gwo-Yu Scientific Reports, Vol. 10, Issue 1 https://doi.org/10.1038/s41598-020-59711-y	journal	February 2020
Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5 Nam, Sung-Eun; Cheung, Yiu Wing Sunny; Nguyen, Thanh Ngoc Communications Biology, Vol. 4, Issue 1 https://doi.org/10.1038/s42003-021-01830-x	journal	March 2021
Mechanisms of resistance to aminoglycoside antibiotics: overview and perspectives Garneau-Tsodikova, Sylvie; Labby, Kristin J. MedChemComm, Vol. 7, Issue 1 https://doi.org/10.1039/C5MD00344J	journal	January 2016
Structure of a TrmA-RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases Alian, A.; Lee, T. T.; Griner, S. L. Proceedings of the National Academy of Sciences, Vol. 105, Issue 19 https://doi.org/10.1073/pnas.0802247105	journal	May 2008
Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA Dunkle, J. A.; Vinal, K.; Desai, P. M. Proceedings of the National Academy of Sciences, Vol. 111, Issue 17 https://doi.org/10.1073/pnas.1402789111	journal	April 2014
A Novel Motif for S -Adenosyl-l-methionine Binding by the Ribosomal RNA Methyltransferase TlyA from Mycobacterium tuberculosis Witek, Marta A.; Kuiper, Emily G.; Minten, Elizabeth Journal of Biological Chemistry, Vol. 292, Issue 5 https://doi.org/10.1074/jbc.M116.752659	journal	December 2016
Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences Li, W.; Godzik, A. Bioinformatics, Vol. 22, Issue 13 https://doi.org/10.1093/bioinformatics/btl158	journal	May 2006
Activity of aminoglycosides, including ACHN-490, against carbapenem-resistant Enterobacteriaceae isolates Livermore, D. M.; Mushtaq, S.; Warner, M. Journal of Antimicrobial Chemotherapy, Vol. 66, Issue 1 https://doi.org/10.1093/jac/dkq408	journal	November 2010
MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0 Tamura, Koichiro; Stecher, Glen; Peterson, Daniel Molecular Biology and Evolution, Vol. 30, Issue 12 https://doi.org/10.1093/molbev/mst197	journal	October 2013
Structural basis for the methylation of A1408 in 16S rRNA by a panaminoglycoside resistance methyltransferase NpmA from a clinical isolate and analysis of the NpmA interactions with the 30S ribosomal subunit Husain, Nilofer; Obranić, Sonja; Koscinski, Lukasz Nucleic Acids Research, Vol. 39, Issue 5 https://doi.org/10.1093/nar/gkq1033	journal	November 2010
Structural basis for the methylation of G1405 in 16S rRNA by aminoglycoside resistance methyltransferase Sgm from an antibiotic producer: a diversity of active sites in m 7 G methyltransferases Husain, Nilofer; Tkaczuk, Karolina L.; Tulsidas, Shenoy Rajesh Nucleic Acids Research, Vol. 38, Issue 12 https://doi.org/10.1093/nar/gkq122	journal	February 2010
Structural insights into the function of aminoglycoside-resistance A1408 16S rRNA methyltransferases from antibiotic-producing and human pathogenic bacteria Macmaster, Rachel; Zelinskaya, Natalia; Savic, Miloje Nucleic Acids Research, Vol. 38, Issue 21 https://doi.org/10.1093/nar/gkq627	journal	July 2010
Functional dichotomy in the 16S rRNA (m ¹ A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization Witek, Marta A.; Conn, Graeme L. Nucleic Acids Research, Vol. 44, Issue 1 https://doi.org/10.1093/nar/gkv1306	journal	November 2015
geopandas/geopandas: v0.8.1 Jordahl, Kelsey; Van den Bossche, Joris; Fleischmann, Martin https://doi.org/10.1101/020917	posted_content	July 2020
XDS Kabsch, Wolfgang Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2 https://doi.org/10.1107/S0907444909047337	journal	January 2010
PHENIX: a comprehensive Python-based system for macromolecular structure solution Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221 https://doi.org/10.1107/S0907444909052925	journal	January 2010
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
The PDB_REDO server for macromolecular structure model optimization Joosten, Robbie P.; Long, Fei; Murshudov, Garib N. IUCrJ, Vol. 1, Issue 4 https://doi.org/10.1107/S2052252514009324	journal	May 2014
cisTEM : user-friendly software for single-particle image processing Grigorieff, Nikolaus; Grant, Tim; Rohou, Alexis Acta Crystallographica Section A Foundations and Advances, Vol. 73, Issue a2 https://doi.org/10.1107/s2053273317082079	journal	December 2017
Structural insight into the methyltransfer mechanism of the bifunctional Trm5 Wang, Caiyan; Jia, Qian; Zeng, Jianhua Science Advances, Vol. 3, Issue 12 https://doi.org/10.1126/sciadv.1700195	journal	December 2017
The structure of the human mitochondrial ribosome Amunts, Alexey; Brown, Alan; Toots, Jaan Science, Vol. 348, Issue 6230 https://doi.org/10.1126/science.aaa1193	journal	April 2015
The complete structure of the 55S mammalian mitochondrial ribosome Greber, B. J.; Bieri, P.; Leibundgut, M. Science, Vol. 348, Issue 6232 https://doi.org/10.1126/science.aaa3872	journal	April 2015
30S Subunit-Dependent Activation of the Sorangium cellulosum So ce56 Aminoglycoside Resistance-Conferring 16S rRNA Methyltransferase Kmr Savic, Miloje; Sunita, S.; Zelinskaya, Natalia Antimicrobial Agents and Chemotherapy, Vol. 59, Issue 5 https://doi.org/10.1128/AAC.00056-15	journal	March 2015
Substrate Recognition and Modification by a Pathogen-Associated Aminoglycoside Resistance 16S rRNA Methyltransferase Vinal, Kellie; Conn, Graeme L. Antimicrobial Agents and Chemotherapy, Vol. 61, Issue 5 https://doi.org/10.1128/AAC.00077-17	journal	March 2017
Synthesis and Spectrum of the Neoglycoside ACHN-490 Aggen, J. B.; Armstrong, E. S.; Goldblum, A. A. Antimicrobial Agents and Chemotherapy, Vol. 54, Issue 11 https://doi.org/10.1128/AAC.00572-10	journal	August 2010
Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides Wachino, J. -i.; Shibayama, K.; Kurokawa, H. Antimicrobial Agents and Chemotherapy, Vol. 51, Issue 12 https://doi.org/10.1128/AAC.00926-07	journal	September 2007
Heterologous Expression and Functional Characterization of the Exogenously Acquired Aminoglycoside Resistance Methyltransferases RmtD, RmtD2, and RmtG Corrêa, Laís L.; Witek, Marta A.; Zelinskaya, Natalia Antimicrobial Agents and Chemotherapy, Vol. 60, Issue 1 https://doi.org/10.1128/AAC.02482-15	journal	November 2015
Critical Residues for Cofactor Binding and Catalytic Activity in the Aminoglycoside Resistance Methyltransferase Sgm Savic, M.; Ilic-Tomic, T.; Macmaster, R. Journal of Bacteriology, Vol. 190, Issue 17 https://doi.org/10.1128/JB.00076-08	journal	June 2008
Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design Lin, Jinzhong; Zhou, Dejian; Steitz, Thomas A. Annual Review of Biochemistry, Vol. 87, Issue 1 https://doi.org/10.1146/annurev-biochem-062917-011942	journal	June 2018
How Antibiotic-Producing Organisms Avoid Suicide Cundliffe, E. Annual Review of Microbiology, Vol. 43, Issue 1 https://doi.org/10.1146/annurev.mi.43.100189.001231	journal	October 1989
PHENIX: a comprehensive Python-based system for macromolecular structure solution. Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor Apollo - University of Cambridge Repository https://doi.org/10.17863/cam.45787	text	January 2010
The complete structure of the 55S mammalian mitochondrial ribosome Greber, Basil J.; Bieri, Philipp; Leibundgut, Marc ETH Zurich https://doi.org/10.3929/ethz-b-000100390	text	January 2015
Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens Matsushita, Takahiko; Sati, Girish C.; Kondasinghe, Nuwan American Chemical Society (ACS) https://doi.org/10.5167/uzh-177583	text	January 2019
cisTEM, user-friendly software for single-particle image processing Grant, Timothy; Rohou, Alexis; Grigorieff, Nikolaus eLife, Vol. 7 https://doi.org/10.7554/eLife.35383	journal	March 2018

Similar Records

30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC

Journal Article · Sun Jun 11 20:00:00 EDT 2023 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:2424139

Functional dichotomy in the 16S rRNA (m¹ A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Journal Article · Mon Nov 23 19:00:00 EST 2015 · Nucleic Acids Research · OSTI ID:1237755

Structural Basis for the Methylation of G1405 in 16S rRNA by Aminoglycoside Resistance Methyltransferase Sgm from an Antibiotic Producer: a Diversity of Active Sites in m7G Methyltransferases

Journal Article · Thu Dec 31 23:00:00 EST 2009 · Nucleic Acids Research · OSTI ID:1019972

Related Subjects

(ribosomal RNA) ribosome RNA methylation
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
RNA methyltransferase
RmtC
aminoglycoside antibiotics
antibiotic resistance
m1A1408
m7G1405
ribosomal ribonucleic acid (rRNA)

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

Citation Formats

References (59)

Similar Records

Related Subjects