Self-remodeling protein complexes are emerging tools for synthetic biology, with applications ranging from renewable chemical production to biomaterial design. Such complexes rely on modular protein binding switches, which alter binding behavior in response to an environmental trigger. However, such technologies remain largely undeveloped due to a dearth of reconfigurable protein domains with optimal dynamic range and sensitivity for smart protein complex design. A promising source from which to draw modular protein binding switches are the cellulosomes of anaerobic fungi, which noncovalently tether enzymes together via modular, protein-binding dockerin domains. Fungal dockerins can be grafted onto a wide variety of enzymes and recombinantly produced in many model microbes, but these cellulosome parts are unexplored as engineering tools because their native assembly mechanism is difficult to replicate in model systems.<br/>Here, we are engineering fungal cellulosome subunits into a general platform for stimuli-responsive protein complex design. We focus on developing pH-responsive assembly as an engineering objective with biological relevance. To make fungal cellulosome parts more amenable to synthetic biology, we generated a dockerin-binding nanobody with nanomolar affinity with which to construct tunable protein complexes. Combining computational molecular modeling with experimental protein characterization, we map key regions in the protein-protein interaction interface and employ this structural knowledge to construct a high throughput screening library from which to select via FACS nanobody-dockerin binding pairs that exhibit different pH-dependent binding behaviors. Towards incorporating these nanobody-dockerin pairs into higher order complexes that remodel in response to pH, we also use molecular simulations to predict how different tethered nanobody configurations perform as central scaffolds for colocalizing multiple dockerin-containing proteins. Overall, this work showcases the potential of untapped fungal cellulosomes as synthetic biology tools and highlights the power of combining a modeling and high-throughput screening approach to protein engineering.

Engineering stimuli-responsive protein complexes inspired by fungal cellulosomes

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.