Native tandem and ion mobility mass spectrometry highlight structural and modular similarities in CRISPR-associated protein complexes from Escherichia coli and Pseudomonas aeruginosa

E. van Duijn, I.M. Barbu, A. Barendregt, M.M. Jore, B. Wiedenheft, M. Lundgren, E.R. Westra, S.J.J. Brouns, J.A. Doudna, J. van der Oost, A.J.R. Heck

Research output: Contribution to journalArticleAcademicpeer-review

72 Citations (Scopus)

Abstract

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 (crRNA). Recently, a cryo-electron microscopy structure of Cascade revealed that the crRNA 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 (IMMS) 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
Original languageEnglish
Pages (from-to)1430-1441
JournalMolecular and Cellular Proteomics
Volume11
DOIs
Publication statusPublished - 2012

Keywords

  • bacterial immune-system
  • crispr-cas systems
  • gas-phase
  • subunit architecture
  • antiviral defense
  • small rna
  • dna
  • recognition
  • prokaryotes
  • sequence

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