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

• DOI: 10.1074/mcp.m112.020263
• Publication Date: 2012-08-24T02:13:48Z
• Authors: Esther van Duijn; Ioana M. Barbu; Arjan Barendregt; Matthijs M. Jore; Blake Wiedenheft; Magnus Lundgren; Edze R. Westra; Stan J.J. Brouns; Jennifer A. Doudna; John van der Oost; Albert J.R. Heck
• Keywords: Models, Molecular; 0301 basic medicine; Biochemistry & Molecular Biology; Protein Stability; 1.1 Normal biological development and functioning; Inverted Repeat Sequences; Molecular; Analytical Chemistry; 3. Good health; Protein Subunits; 03 medical and health sciences; Bacterial Proteins; Underpinning research; Models; Tandem Mass Spectrometry; Multiprotein Complexes; Chemical Sciences; Pseudomonas aeruginosa; Genetics; Escherichia coli; Generic health relevance; Infection; Protein Binding

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.