Waghmare, S. | bioRxiv Science

bioRxiv2026

Exploring differential interactional preferences of enzyme-bearing dockarins for cohesin domains in the Clostridium thermocellum cellulosome

Using its 9 sequentially-placed cohesin (Coh) domains as bait, each CipA scaffoldin chain in a Clostridium thermocellum cellulosome binds to (and displays) some combination of 9 out of over [~]70 available dockerin (Doc) domain-bearing lignocellulose-degrading enzymes, at any time. Domains numbered Coh3 through Coh8 show over 94 % pairwise sequence identity with each other, but only [~] 61-76 % identity with Coh1, Coh2, and Coh9. Such identities are much lower ([~] 40-60 %) amongst Doc domains; however, amongst both Coh and Doc domains, polypeptide backbone folds are highly conserved, suggesting that loading preferences of enzyme-bearing Doc domains upon Coh domains must depend upon their relative abundances, and pairwise affinities. To explore this further, we used microscale thermophoresis (MST), size exclusion chromatography (SEC), native polyacrylamide gel electrophoresis (NPGE), mass spectrometry (MS) and bioinformatics-based approaches (BIBA), to examine 28 Coh-Doc pairwise interactions involving recombinant Coh [Coh1, Coh2, Coh3, Coh9] and enzyme-bearing Doc [Cel8A, Cel9F, Man26/5H, Cel9R, Xyn10C, Xyn11D, Xyn10Z] domains. Interactions were found to occur with varying affinities, suggesting that Coh1 prefers Xyn11D; Coh2 prefers Xyn10C; Coh3 prefers Cel9R; Coh9 prefers Cel9R; Coh1/Coh2 prefer Xyn partners; Coh3/Coh9 prefer Cel partners. Dual modes of binding are shown by Coh1 with Xyn10C and Xyn11D; Coh2 with Xyn10Z, Cel8A, and Cel9R; Coh3 with Cel8A, and Cel9R; and Coh9 with Xyn10C, suggesting that Doc domains use either of their two homologous helices (1 and 3) to bind to Coh domains, as earlier proposed. ImportanceBacteria such as Clostridium thermocellum use extracellular enzyme complexes called cellulosomes to degrade and use cellulose. Each complex uses a linear chain of nine cohesin (Coh) domains called a scaffoldin to bind to (and display) any nine of over seventy available xylan or cellulose-degrading enzymes that bear dockerin (Doc) domains. Understanding interactions between Coh and Doc domains facilitates an appreciation of how cellulosomes are assembled and supports the building of protein-engineered constructs that utilize such interactions for many conceivable enzymatic and other applications. The significance of the presented research lies in its demonstration of the differential modes and pairwise affinities of different Coh-Doc interactions, using recombinant protein constructs and a combination of quantitative, semi-quantitative and qualitative analytical methods.

biochemistry↗

bioRxiv2018

Identification of RNA-associated peptides, iRAP, defines precise sites of protein-RNA interaction

ABSRACTThe identification of ever-increasing numbers of RNA species has underlined the importance of robust characterization of bona fide sites of protein-RNA interaction. UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. Here we sought to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, identification of RNA-associated peptides (iRAP), was used to locate 1331 unique RNA-interaction sites at single amino acid residue resolution in 324 S. cerevisiae proteins. Our identified RNA-interaction sites in characterized RNA-protein complex agree well with available high-resolution structures. In known RNA-interacting protiens, 317 sites fall into known and suspected RNA-interaction domains while only 21 sites fall into other annotated sequence features. Strikingly, 993 of the sites identified fall into protein regions that lack any recognizable protein domain structure or annotated sequence features. This suggests that, despite binding RNA in vivo, many of these proteins will not have defined functions in RNA metabolism.\n\nHighlights Method identifies precise RNA-binding sites across proteome\n1331 sites identified that contact RNA in 324 proteins in S. cerevisiae\nRNA binding takes place predominantly outside known protein domains

molecular biology↗