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

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. 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. Molecular & Cellular

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

“…1A) will cofunction with the Cst complex as the effector nuclease/helicase. Based on expectations from other Type I systems, the Cst crRNP may function as a surveillance complex that recruits Cas3 nuclease to cleave invader DNA (Brouns et al 2008;Cady and O'Toole 2011;Jore et al 2011;Wiedenheft et al 2011a,b;Nam et al 2012;Sashital et al 2012;van Duijn et al 2012;Westra et al 2012b;Mulepati and Bailey 2013;Sinkunas et al 2013;Hochstrasser et al 2014;Huo et al 2014). We likely did not detect Cas3 in our isolated Cst crRNP complexes (Fig.…”

“…1A). While the Csa and Cst complexes have not been extensively studied, Cas5, Cas7, Cas8 (large subunit), and Cas11 (small subunit) superfamily proteins comprise other characterized Type I crRNPs (the latter sometimes fused to the large subunit as a domain) (Brouns et al 2008;Lintner et al 2011b;Wiedenheft et al 2011a,b;Nam et al 2012;Plagens et al 2012;van Duijn et al 2012;Brendel et al 2014). The signature Cas3 (HD nuclease-DExH helicase) proteins of Type I systems interact with the crRNPs and cleave invader DNA (Cady and O'Toole 2011;Westra et al 2012a;Mulepati and Bailey 2013;Sinkunas et al 2013;Hochstrasser et al 2014;Plagens et al 2014).…”

Analytical Approaches for Size and Mass Analysis of Large Protein Assemblies

“…In this defense mechanism, a protein-RNA complex plays a pivotal role, termed cascade in Escherichia coli, which targets RNA molecules encoded in the genome of the bacteria toward viral DNA, where, upon binding, it hampers viral replication, thereby suppressing infection. The stoichiometry and structures of a variety of such CRISPR-related complexes, which typically contain half a dozen different proteins and a crRNA sequence, have recently been elucidated with the aid of native MS by detailing the stoichiometry, demonstrating subunit connectivity with solution-phase dissociation, and determining subunit arrangement and shape from IMMS (see Figure 4) (81)(82)(83). The resulting models of the CRISPR-related complexes were shown to be in excellent agreement with cryoEM reconstructions of the same complexes and a critical first step in assigning those EM densities.…”

Ion Mobility-Mass Spectrometry Reveals Highly-Compact Intermediates in the Collision Induced Dissociation of Charge-Reduced Protein Complexes

“…The accuracy of the IM-MS measurements, as well as the number of subcomplexes observed during data collection directly influences the confidence level and information content of the models generated [10][11][12]. Such IM-MS methods have recently been used to construct models of polydisperse heatshock proteins [13], ATP synthase [14], the CRISPR-associated cascade assembly [15], amyloid-associated aggregates [16], and replisome-related complexes [17].…”