Integrative SAXS and AFM analysis of engineered carbohydrate-active enzyme assemblies with tunable spatial...

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Protein Engineering

Background:

• Cellulosomes are complex, multi-enzyme structures crucial for biomass degradation.
• Their catalytic efficiency is highly dependent on the precise spatial arrangement of constituent enzymes.
• The inherent conformational flexibility of cellulosomes has historically hindered quantitative structural analysis.

Purpose of the Study:

• To establish a methodological framework for designing and analyzing constrained multi-enzyme complexes.
• To overcome limitations in characterizing the topology and inter-enzyme distances in flexible protein assemblies.
• To enable the generation of defined multi-enzyme constructs for structural and functional studies.

Main Methods:

• Utilized the Jo-In scaffold to fix the positions of specific enzymes (endoglucanases AtCel8A, AtCel9R, and xylanase AtXyn11A).
• Employed small-angle X-ray scattering (SAXS) for analyzing the overall compaction and architecture of the assemblies.
• Integrated atomic force microscopy (AFM) for single-particle level validation of SAXS-derived models.
• Performed atomistic modeling using DADIMODO and BILBO-MD for consistent structural organization analysis.

Main Results:

• Generated defined multi-enzyme complexes with characterized architectures.
• SAXS analysis revealed distinct compaction profiles for different enzyme arrangements in two-glucanase complexes.
• AFM imaging corroborated SAXS findings, validating the models at the single-particle level.
• SAXS measurements of three-enzyme assemblies showed consistent radii of gyration (53 ± 2 Å) and maximum dimensions (180–200 Å).
• Atomistic modeling converged on consistent average spatial organizations and interdomain distance ranges.

Conclusions:

• The Jo-In scaffold provides a versatile tool for creating and structurally analyzing tailored multi-enzyme assemblies.
• This quantitative framework advances the understanding of structure-function relationships in modular, dynamic protein complexes.
• The methodology enables precise control over enzyme spatial organization, crucial for optimizing catalytic efficiency.