Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility

Modular biosynthesis of small molecules—where enzyme units can be swapped in and out of assembly line complexes to produce desired products—is a distant goal in the lab despite a huge diversity of modular systems in nature. Part of the challenge is in understanding how modules interact and hand off intermediates. Reimer et al. determined crystal structures of portions of a nonribosomal peptide synthetase, including a full dimodule. Module positioning differed between these structures even when the same intermediate was attached to the enzyme. The authors used small-angle x-ray scattering to confirm that large conformational changes are possible during biosynthesis and handoff between modules.Science, this issue p. eaaw4388INTRODUCTIONNonribosomal peptide synthetases (NRPSs) are microbial megaenzymes that make a wide variety of small-molecule products, including many that are clinically used as antitumors, antibiotics, or immunosuppressants. Nonribosomal peptide synthesis proceeds with assembly-line logic, where each station on the NRPS assembly line is a multidomain unit called a module. An excellent understanding of the structures and activities of isolated modules has been established, but much less is known about how modules work with each other in the context of the larger NRPS. Structural investigation of multimodular NRPSs is needed to understand NRPS architecture, organization, and intramodular function during the synthetic cycle of an NRPS and to facilitate the longstanding goal of bioengineering for production of new-to-nature bioactive small molecules.RATIONALETo gain insight into outstanding trans- and supermodular questions in NRPS function, we performed x-ray crystallography with a series of constructs of the dimodular NRPS protein linear gramicidin synthetase subunit A (LgrA). We performed complementary small-angle x-ray scattering experiments to analyze the behavior of the NRPS in solution. We also performed direct coupling analysis to confirm the biological relevance and evolutionary conservation of observed interdomain interfaces. Both the structures and direct coupling analyses were used to guide mutagenesis studies designed to enhance the activity of a chimeric NRPS.RESULTSWe have determined five independent crystal structures of constructs of LgrA, bound with a series of ligands and intermediate analogs, to resolutions between 2.2 and 6 Å. The crystallized constructs include the complete initiation module and from one to all three canonical domains from the elongation module. Some structures are in markedly different conformations, inferring large movements, and each structure seems to be in a catalytically relevant state. Small-angle x-ray scattering indicates that LgrA is also very flexible in solution, confirming that markedly different conformations are a bona fide feature of NRPS biology. The structures reveal previously unobserved states, including a full condensation conformation, where the thiolation (T) domains from both the initiation and elongation modules are simultaneously bound at the condensation (C) domain. Similar conformations in high-resolution structures allow analyses of the productive T:C domain-domain interface, which mediates the only known functional link between modules. Direct coupling analysis applied to large collections of NRPS sequences provides strong support for the biological relevance and evolutionary conservation of observed interdomain interfaces. Furthermore, both the structures and coupling scores for mutational effects were used to guide bioengineering, and we were able to double the activity of a module-swapped chimeric NRPS by introducing two point mutations at the unnatural T:C domain-domain interface.CONCLUSIONThe structures and small-angle x-ray scattering show NRPSs undergo very large conformational changes and challenge the general assumption that NRPSs have regular higher-order architecture. They demonstrate that there is no strict coupling between the catalytic state of a particular module and the overall conformation of the multimodular NRPS and suggest that the T:C interaction for condensation is the only point where adjacent modules must coordinate. This feature can be exploited in module-swapping bioengineering to produce new useful nonribosomal peptides.Structures of a dimodular NRPS protein reveal the central condensation state and infer very large conformational changes.A series of crystal structures of the dimodular nonribosomal peptide synthetase protein LgrA includes a structure of the condensation state (left). Condensation is the central event in synthesis, elongating the peptide intermediate and passing it to the downstream module. Additional structures in condensation and thiolation states show large conformational differences (indicated by arrows), which are supported by solution small-angle x-ray scattering data. These structures show decoupling of the catalytic state and overall conformation and imply that coordination of adjacent modules’ catalytic states is only required at condensation. The structures and coevolution analyses enable improvement of activity of a module-swapped chimeric enzyme (bottom left).Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.