Structural insights into the domain interactions and conformational changes of the non-ribosomal peptide synthetases and firefly luciferase
Sundlov, Jesse Adam
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Many microorganisms employ a ribosome free pathway in the synthesis of complex peptide-based natural products that frequently have important bioactivities, including antibiotic, anticancer, and immunosuppressant properties. There has been a tremendous effort to detail how these non-ribosomal peptide synthetases (NRPSs) catalyze such complex and elegant reactions. In these times of increasing antibiotic resistance with no corresponding response in the development of new drugs, a thorough understanding of how these assembly lines operate not only satiates our scientific curiosity, but would allow for the more efficient design of novel bioactive compounds. Acting in an assembly line-like manner, an NRPS system consists of multiple modules, with each module being responsible for the recognition and incorporation of one amino acid into a growing peptide intermediate. An individual NRPS module consists of multiple catalytic domains, and the entire system can be expressed as one polypeptide chain or spread over several distinct proteins. Individual structures of the core NRPS domains have been elucidated, while the structure of an entire module was published in 2008. This structure demonstrated one catalytic conformation, and suggested that large-scale inter-domain rearrangements must occur in order to accommodate all necessary catalytic reactions. The objective of the work presented herein was to determine the inter-domain interactions of an A-PCP multidomain NRPS, with the hypothesis that a large scale domain rotation previously demonstrated in the related acyl-CoA synthetases would apply to NRPS adenylation domains during substrate transfer to the PCP domain. The EntE-B structure presented in Chapter Two confirms this conformation and demonstrates the interface between these two core NRPS domains, while also revealing a fully-occupied binding pocket and pantetheine tunnel that captured the pose of all players in the covalent transfer of substrate to carrier protein. Analysis of the EntE-B structure led to bioinformatic studies that revealed unique characteristics of the A-PCP linker regions – particularly the conservation of multiple proline residues. Mutagenesis experiments confirmed the importance of these conserved residues in product formation. The results, detailed in Chapter Three, suggest an elevated structural role for these inter-domain regions beyond a simple tethering of domains. Finally, the catalytically-essential domain rotation first revealed in the acyl-CoA synthetases, then demonstrated in NRPSs with the EntE-B structure, has been shown for the first time in firefly luciferases, and is detailed in Chapter Four. This novel structure suggests that the widely accepted luciferase catalytic mechanism needs to be altered, and we put forth an alternate mechanism based on the newly obtained structural information.