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

• Published in: Structure (London) Vol. 25; no. 5; pp. 783 - 793.e4
• Main Authors: Tarry, Michael J., Haque, Asfarul S., Bui, Khanh Huy, Schmeing, T. Martin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 02.05.2017
• Subjects: adenosine-vinylsulfonamide inhibitor; adenylation activity; Bacteria - drug effects; Bacteria - enzymology; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Binding Sites; conformational flexibility; Enzyme Inhibitors - pharmacology; MbtH-like protein; mega-enzyme; nonribosomal peptide synthetase; NRPS; Peptide Synthases - antagonists & inhibitors; Peptide Synthases - chemistry; Peptide Synthases - metabolism; Protein Binding; single-particle electron microscopy; Sulfonamides - pharmacology; synthetic cycle; Vinyl Compounds - pharmacology; X-ray crystallography; X-ray crystallography; nonribosomal peptide synthetase; MbtH-like protein; NRPS; adenosine-vinylsulfonamide inhibitor; mega-enzyme; conformational flexibility; synthetic cycle; adenylation activity; single-particle electron microscopy
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2017.03.014

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. [Display omitted] •Structure of cross-module NRPS DhbF construct bound to an MbtH-like protein (MLP)•Adenosine-vinylsulfonamide inhibitor helped capture protein in a new conformation•MLP binds with nanomolar affinity and is required for substrate and inhibitor binding•Multi-modular DhbF exhibits large conformational variability between modules Tarry et al. report the crystal structure of a cross-modular tri-domain of the NRPS DhbF. The protein adopts a novel conformation with limited cross-module contacts. EM reconstructions of dimodular DhbF show large variations in the supermodular configuration. These data suggest that NRPSs lack uniform, higher-order architecture.