Protein-protein cross-linking in the C. thermocellum Cascade complex

The goal of this investigation was to characterize protein-protein interactions within the Cas proteins in the Type I-B Cascade complex from C. thermocellum. The project sought to evaluate the feasibility of chemical protein-protein cross-linking for the investigation of purified crRNP complexes. Although there is not enough protein-protein interaction data available to validate the results of this study, the information available from the common architectural features of the Type I crRNP complexes was used to explain the outcome from protein-protein cross-linking study.

Chemical cross-linking was performed using BS3, which targets primary amines in the side chains of lysine residues on protein surface as well as protein N-termini. In order to optimize the cross-linker:protein ratio to be used for cross-linking, the purified Cascade complex was incubated with increasing molar excesses of BS3 cross-linker in the cross-linker to protein ratio 5:1, 10:1, 25:1, 50:1 , 100:1 and 200:1. The results of this titration were analyzed with

SDS-3. RESULTS 105 PAGE as shown in Figure 3.15. The details of the cross-linking reaction are described under Section 2.2.7.

Figure 3.15 Analysis of protein-protein cross-linking by SDS-PAGE.

Coomassie stained SDS-PAGE gel showing the results of protein-protein cross-linking performed in the Cascade complex using different concentration of BS3 cross-linker. Lane 1: The purified Cascade complex alone (without any cross-linker) consisted of Cas7 (35 kDa), Cas5 (28 kDa), Cas6 (26 kDa) and two fragments for Cas8b the larger fragment 72 kDa and a smaller ~15 kDa fragment Cas8b*. Lane 2-7: Optimization of cross-linker to protein complex ratio. As the molar excess of BS3 over Cascade increases, the intensity of bands corresponding to Cas proteins diminishes. At the same time bands corresponding to protein-protein cross-links appear with even 5:1 cross-linker to protein ratio. The ratio 100:1 and later corresponds to high molecular aggregates which can be neglected. lM:

Protein marker (BioRad).

Upon the SDS-PAGE analysis it was observed that the purified Cascade complex from C.

thermocellum comprised four Cas proteins Cas7, Cas5, Cas6 and Cas8b (Figure 3.15 Lane 1).

Throughout the titration the protein-protein cross-links could be observed in all the cases where even a minimal amount (5 molar excess) of BS3 cross-linker was present. With increasing amounts of cross-linker the band corresponding to protein-protein cross-links also enhanced (Figure 3.15 Lane 2-7). At very high cross-linker amounts there were higher order aggregates and artefacts appearing as a smear at high molecular weight (Figure 3.15 Lane 6 and 7).

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For the MS analysis the cross-linker to protein ratio of 75:1 was selected as the optimal ratio for the final cross-linking experiment. This ratio was selected to avoid the complex high-order aggregates and at the same time to get enough protein-protein cross-links, for the MS analysis.

The cross-linked complexes were hydrolyzed with trypsin and the resulting peptide mixture was further enriched for cross-linked peptides using size exclusion chromatography (Figure 1.13), details in section 2.2.7.3.

The enriched cross-links were analyzed by LC-MS/MS in DDA mode on an Orbitrap Fusion instrument and the cross-linked peptides were identified using pLink, using 1% FDR and the results are presented in a cross-link map (Figure 3.16) after applying a score cut-off of 2.0 (based on the p-value). The spectra for the cross-links identified were also manually checked to confirm the assigned cross-link. Further the cross-link species such as mono-links or loop links (Figure 1.13) were filtered out from the final list of cross-links, as they carry limited information not significant in the investigation of protein-protein interactions. In the end only unique inter-protein and intra- inter-protein cross-links are reported in this work.

3.3.2.1 Inter-protein cross-links identified for Cas5, Cas6, Cas7 and Cas8b proteins

From the MS analysis 126 unique inter-protein cross-links shown in Figure 3.16 were identified within the four Cas proteins (Appendix, Table 6.2). However due to a lack of structures available for these proteins, it was not possible to map the cross-link sites on three dimensional structures. Nonetheless, the cross-links identified in the Cas proteins were in agreement to the Cas protein stoichiometry and the position of a particular Cas protein in the Type I Cascade complexes [22], as determined in recent studies on the architecture and organization of E. coli Cascade complex [105, 106].

Two inter-protein cross-links were identified for the Cas6 protein, K²² was observed cross-linked to K¹⁵⁵ in the N-terminal region of Cas7 protein and K¹⁰⁶ was observed cross-linked to K⁷³ in the N-terminal region of Cas8b. The Cas6 protein forms the head of an assembled Cascade complex interacting with the 3’ end of crRNA, as reported earlier in the Type I-E E. coli Cascade complex.

Further, the head protein interacts with the first Cas7 protein that forms the backbone of a Cascade complex and also the large subunit Cas8b which spans the entire length of the complex. In addition no cross-link between Cas6 and Cas5 (the tail protein) was observed, which is in agreement with the positioning of Cas5 and Cas6 proteins in the Cascade complex as

3. RESULTS 107 shown in (Figure 1.4 and 1.15) i.e., the head and tail proteins are distant enough for cross-linking via BS3 to occur.

Figure 3.16 Protein-protein cross-linking map for the C. thermocellum Cascade complex.

A protein-protein cross-link map made using xiNET. Only the inter-protein cross-links identified for Cas6, Cas5, Cas7 and Cas8b proteins are shown. The cross-links were identified using pLink with 1% FDR. The cross-links indicated are the one remaining after applying a score cut off 2.0. The gray shaded regions indicate high density of cross-links between those regions. The blue shaded regions indicate lysine rich regions of the proteins with a high density of cross-links observed for the lysine residues. Further details of all the identified cross-links are provided in the Appendix (Table 6.2)

In Cas5 a higher number of cross-links were identified. There were 12 unique cross-links identified between Cas5 and Cas8b and 37 between Cas5 and Cas7. Most of these cross-links were confined to a lysine rich region in Cas5 with four residues K⁷², K⁸⁴, K⁸⁵ and K⁹⁷ as shown in Figure 3.16. Cas5 is a part of the tail assembly in Cascade complex [105] which comprises Cas5, Cas7 and in this case Cas8b subunits.

Between Cas7 and Cas8b proteins 75 unique cross-links were identified corresponding to lysine residues distributed throughout the protein sequence. In both Cas7 and Cas8b a very high number of cross-links were identified in the two lysine rich regions of both the proteins as

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shown in Figure 3.16. The high number of cross-links identified is in agreement with the stoichiometry determined. Both the proteins are present in more than one copy; therefore the cross-links identified cannot be assigned to any one subunit of the two proteins. Rather it can only be speculated that the regions with high density of cross-links are in close spatial proximity, likely to form cross-links in the presence of a chemical cross-linker such as BS3. In addition, the Cas7 protein has been reported to form the helical backbone of the Cascade-like complexes and is supported by the large subunit Cas8b throughout the length of the complex (Figure 1.4), therefore so many protein-protein interactions between the two proteins are likely to occur.

3.3.2.2 Intra-protein cross-links identified for Cas5, Cas6, Cas7 and Cas8b proteins

In addition to the inter-protein cross-links described above, a large number of intra-protein cross-links were also identified for all the four Cas proteins. A total of 16 unique intra-protein cross-links were observed in Cas5 and six in Cas6 (Appendix, Table 6.3 and 6.4). Only for these two proteins Cas5 and Cas6 protein which are present in a single copy in the Cascade complex, the identified cross-links could be mapped unambiguously on the protein sequence (Appendix, Figure 6.6).

In Cas7 there were 112 unique intra-protein cross-links and for Cas8b there were 230 unique intra-protein cross-links. However these numbers cannot be assigned to any one subunit of the two proteins in particular. Considering the stoichiometry of the two proteins, there is a high ambiguity in mapping these cross-link sites to the protein sequence and assigning them to different protein subunits. Hence further details of the intra-protein cross-links identified for Cas7 and Cas8b are not included in this work.

These protein-protein cross-linking studies provide the first experimental evidence for the protein-protein interactions in Type I-B Cascade complex from C. thermocellum and can be used in the development of a structural model.