Molecular Mechanisms Remodeling Dynamic Landscapes in Multidomain Enzymatic Factories

Project Summary Our laboratory is broadly dedicated to understanding the role of protein dynamics while facilitating the engineering of nonribosomal peptide synthetases (NRPSs). Recent structure determination advancements have highlighted that multidomain proteins are often dynamic, exhibiting distinct functional outputs mediated by specific domain interactions. Elucidating the mechanisms underlying how domains transition between these stable interactions remains an outstanding question that must be answered for effectively engineering dynamic multidomain proteins like NRPSs. NRPSs utilize a modular, multidomain architecture to covalently link simple substrates and synthesize complex natural products, many with therapeutic properties (e.g., antibiotics like bacitracin or antitumor agents like epothilones). Swapping domains or modules within NRPSs holds promise for developing improved pharmaceuticals. However, their inherent dynamic nature complicates the interpretation of mutagenesis data obtained through traditional static structures. Engineering NRPSs is thus akin to solving a complex, multidimensional puzzle. To address this challenge, we will combine nuclear magnetic resonance (NMR) spectroscopy and computational approaches to determine structural ensembles that enable the prediction of how mutations affect protein functions by altering their dynamics. Furthermore, we will initiate single-molecule studies to link these intra-domain changes to inter-domain communication, enabling a holistic interpretation of product formation assays. Our results will identify key residues that must be modified to alter substrate and domain recognitions and also to maintain functional dynamics during NRPS engineering. Leveraging over 20 years of expertise in NMR protein dynamics and large protein studies, we will collaborate with specialists in all other relevant areas to ensure the success of this research. Our findings will pave the way for effective NRPS engineering, facilitating the development of novel therapeutics. Additionally, our work will offer a valuable paradigm for understanding how dynamic multidomain systems remodel domain interactions to regulate function, a ubiquitous phenomenon across biological systems.