Ligand-mediated changes in conformational dynamics of NpmA: Implications for ribosomal interactions

Nilofer Husain, Nikhil Kumar Tulsian, Wang Loo Chien, Sushant Suresh, Ganesh Srinivasan Anand, J. Sivaraman

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Aminoglycosides are broad-spectrum antibiotics that bind to the 30S ribosomal subunit (30S) of bacteria and disrupt protein translation. NpmA, a structurally well-characterized methyltransferase identified in an E. coli clinical isolate, catalyzes methylation of 30S at A1408 of the 16S rRNA and confers aminoglycoside resistance. Using sucrose cushion centrifugation and isothermal titration calorimetry, we first confirmed the binding between NpmA and 30S. Next, we performed amide Hydrogen/Deuterium Exchange Mass Spectrometry (HDXMS) of apo NpmA and in the presence and absence of SAM/SAH. We observed that ligand binding resulted in time-dependent differences in deuterium exchange not only at the ligand-binding pocket (D25-D55 and A86-E112) but also in distal regions (F62-F82 and Y113-S144) of NpmA. These results provide insights into methylation group donor cofactor-mediated allostery in NpmA in the ligand-bound states, which could not be observed in the static endpoint crystal structures. We predict that the two distal sites in NpmA form part of the allosteric sites that importantly are part of the main 16S rRNA binding interface. Thus HDXMS helped uncover allosteric communication relays that couple SAM/SAH binding sites with the ribosome-binding site. This highlights how HDXMS together with X-ray crystallography can provide important allosteric insights in protein-ligand complexes.

Original languageEnglish
Article number37061
JournalScientific Reports
Volume6
DOIs
Publication statusPublished - 15-11-2016
Externally publishedYes

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Deuterium
Ligands
Hydrogen
Mass Spectrometry
Aminoglycosides
Methylation
Binding Sites
Allosteric Site
Ribosome Subunits
Calorimetry
X Ray Crystallography
Methyltransferases
Protein Biosynthesis
Centrifugation
Ribosomes
Amides
Sucrose
Escherichia coli
Anti-Bacterial Agents
Bacteria

All Science Journal Classification (ASJC) codes

  • General

Cite this

Husain, N., Tulsian, N. K., Chien, W. L., Suresh, S., Anand, G. S., & Sivaraman, J. (2016). Ligand-mediated changes in conformational dynamics of NpmA: Implications for ribosomal interactions. Scientific Reports, 6, [37061]. https://doi.org/10.1038/srep37061
Husain, Nilofer ; Tulsian, Nikhil Kumar ; Chien, Wang Loo ; Suresh, Sushant ; Anand, Ganesh Srinivasan ; Sivaraman, J. / Ligand-mediated changes in conformational dynamics of NpmA : Implications for ribosomal interactions. In: Scientific Reports. 2016 ; Vol. 6.
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Ligand-mediated changes in conformational dynamics of NpmA : Implications for ribosomal interactions. / Husain, Nilofer; Tulsian, Nikhil Kumar; Chien, Wang Loo; Suresh, Sushant; Anand, Ganesh Srinivasan; Sivaraman, J.

In: Scientific Reports, Vol. 6, 37061, 15.11.2016.

Research output: Contribution to journalArticle

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AU - Husain, Nilofer

AU - Tulsian, Nikhil Kumar

AU - Chien, Wang Loo

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AU - Anand, Ganesh Srinivasan

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