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

Abstract

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.