(58b) Self-Remodeling Protein Complexes Inspired By Fungal Cellulosomes

Engineered protein systems that sense different environmental signals to produce specific outputs have broad applications towards developing smart biomaterials or for building programmable biotherapeutics. Parts with which to construct these “molecular computers” that function by altering sets of protein-protein interactions in response to stimuli are a key technology gap not well addressed by current sets of modular, interacting protein domains, which are limited in their generalizability to different protein cargo and their ability to be multiplexed. Fungal cellulosomes, the multi-enzyme complexes produced by anaerobic fungi to degrade plant matter in the guts of herbivores, are an attractive template for designer, stimuli-responsive protein complexes because they scaffold proteins of diverse structure and function using modular parts that are highly engineerable (Figure 1). Cellulosomes assemble via non-covalent interactions between enzyme-fused dockerin domains and cohesin domains repeated on a central scaffoldin protein. However, fungal cellulosome parts have never been explored for engineering applications because the sequence and structure of dockerin’s binding partner, cohesin, remain unknown. Here, we combined molecular modeling with high throughput screening to develop the dockerin and a synthetic cohesin substitute, a nanobody, into a set of protein parts for constructing complexes that remodel in response to pH - a biologically useful signal. We first used modeling tools to predict the binding interface and constructed a yeast surface display screening library in which 22 positions were combinatorially mutated to histidine to impart pH-sensitive binding. Parallel screens produced switches with different binding and switch thermodynamics, providing a dataset with which to generate, via atomistic modeling, mechanistic hypotheses for switch function that will inform the design of new parts with tailored switch-like behavior. Towards synthesizing higher order complexes, we employed molecular dynamics to screen scaffolding protein architectures for designs capable of binding several partners simultaneously, accelerating the development of functional, self-remodeling complexes. Overall, this work showcases the potential for cellulosomes as synthetic biology tools and highlights the power of a combined modeling and high-throughput screening approach in protein engineering.