Structural insights into Escherichia coli phosphopantothenoylcysteine synthetase by native ion mobility–mass spectrometry

Abstract CoaBC, part of the vital coenzyme A biosynthetic pathway in bacteria, has recently been validated as a promising antimicrobial target. In this work, we employed native ion mobility–mass spectrometry to gain structural insights into the phosphopantothenoylcysteine synthetase domain of E. coli CoaBC. Moreover, native mass spectrometry was validated as a screening tool to identify novel inhibitors of this enzyme, highlighting the utility and versatility of this technique both for structural biology and for drug discovery.

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Native Tandem and Ion Mobility Mass Spectrometry Highlight Structural and Modular Similarities in Clustered-Regularly-Interspaced Shot-Palindromic-Repeats (CRISPR)-associated Protein Complexes From Escherichia coli and Pseudomonas aeruginosa

Molecular & Cellular Proteomics ◽

10.1074/mcp.m112.020263 ◽

2012 ◽

Vol 11 (11) ◽

pp. 1430-1441 ◽

Cited By ~ 63

Author(s):

Esther van Duijn ◽

Ioana M. Barbu ◽

Arjan Barendregt ◽

Matthijs M. Jore ◽

Blake Wiedenheft ◽

...

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Ion Mobility ◽

Protein Complexes ◽

Acquired Resistance ◽

Native Mass Spectrometry ◽

Ion Mobility Mass Spectrometry ◽

E Coli ◽

Ribonucleoprotein Complexes ◽

Specific Association

The CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) immune system of bacteria and archaea provides acquired resistance against viruses and plasmids, by a strategy analogous to RNA-interference. Key components of the defense system are ribonucleoprotein complexes, the composition of which appears highly variable in different CRISPR/Cas subtypes. Previous studies combined mass spectrometry, electron microscopy, and small angle x-ray scattering to demonstrate that the E. coli Cascade complex (405 kDa) and the P. aeruginosa Csy-complex (350 kDa) are similar in that they share a central spiral-shaped hexameric structure, flanked by associating proteins and one CRISPR RNA. Recently, a cryo-electron microscopy structure of Cascade revealed that the CRISPR RNA molecule resides in a groove of the hexameric backbone. For both complexes we here describe the use of native mass spectrometry in combination with ion mobility mass spectrometry to assign a stable core surrounded by more loosely associated modules. Via computational modeling subcomplex structures were proposed that relate to the experimental IMMS data. Despite the absence of obvious sequence homology between several subunits, detailed analysis of sub-complexes strongly suggests analogy between subunits of the two complexes. Probing the specific association of E. coli Cascade/crRNA to its complementary DNA target reveals a conformational change. All together these findings provide relevant new information about the potential assembly process of the two CRISPR-associated complexes.

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Metabolic profiling of Escherichia coli by ion mobility-mass spectrometry with MALDI ion source

Journal of Mass Spectrometry ◽

10.1002/jms.1850 ◽

2010 ◽

Vol 45 (12) ◽

pp. 1383-1393 ◽

Cited By ~ 38

Author(s):

Prabha Dwivedi ◽

Geoffery Puzon ◽

Maggie Tam ◽

Denis Langlais ◽

Shelley Jackson ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Ion Mobility ◽

Metabolic Profiling ◽

Ion Source ◽

Ion Mobility Mass Spectrometry

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EM∩IM: software for relating ion mobility mass spectrometry and electron microscopy data

The Analyst ◽

10.1039/c5an01636c ◽

2016 ◽

Vol 141 (1) ◽

pp. 70-75 ◽

Cited By ~ 11

Author(s):

Matteo T. Degiacomi ◽

Justin L. P. Benesch

Keyword(s):

Electron Microscopy ◽

Mass Spectrometry ◽

Structural Biology ◽

Cross Sections ◽

Ion Mobility ◽

Ion Mobility Mass Spectrometry ◽

Density Maps ◽

Transmission Electron ◽

Microscopy Data ◽

Means Of Transmission

EM∩IM enables the calculation of collision cross-sections from electron density maps obtained, for example, by means of transmission electron microscopy. This capability will further aid the integration of ion mobility mass spectrometry with modern structural biology.

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