X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture.

Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture.