Structure
Volume 25, Issue 5, 2 May 2017, Pages 783-793.e4
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Article
X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture

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Highlights

  • 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

Summary

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.

Keywords

nonribosomal peptide synthetase
NRPS
mega-enzyme
synthetic cycle
MbtH-like protein
adenosine-vinylsulfonamide inhibitor
adenylation activity
conformational flexibility
single-particle electron microscopy
X-ray crystallography

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