Integrative SAXS and AFM analysis of engineered carbohydrate‐active enzyme assemblies with tunable spatial organization

Cellulosomes are multi-enzyme assemblies whose catalytic efficiency depends on the spatial organization of their components. However, their pronounced conformational flexibility has precluded quantitative characterization of inter-enzyme distances and overall topology. Here, we present a methodological framework to tailor and analyze the architecture of constrained multi-enzyme complexes composed of two endoglucanases AtCel8A and AtCel9R and one xylanase AtXyn11A by fixing enzyme positions using the Jo-In scaffold. This approach enables generation of defined assemblies structurally characterized by small-angle X-ray scattering (SAXS) and/or atomic force microscopy (AFM). SAXS analysis of the two-glucanase complexes reveals distinct scattering profiles corresponding to different degrees of compaction depending on the enzyme spatial arrangement. Complementary AFM imaging of bifunctional assemblies supports SAXS derived models at the single-particle level, reinforcing the robustness of the proposed workflow. The three-enzyme assemblies' SAXS measurements distinguish different constructions while showing relatively homogeneous radii of gyration (53 ± 2 Å) and maximum dimensions of 180 to 200 Å. Atomistic modeling using two independent approaches, DADIMODO and BILBO-MD, converges toward consistent average spatial organizations with constrained interdomain distance ranges, and consistent quantitative parameters validating the structural models. Altogether, the results establish a quantitative framework for designing tailored multi-enzyme assemblies. Jo-In scaffold provides a versatile tool for structural analysis of modular, dynamic protein complexes, advancing our understanding of structure-function relationship in multi-enzyme systems.