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Living cells are constantly producing proteins (polypeptides) by translating genetic sequences (messenger RNA) using the large molecular complex called the ribosome. Surprisingly, cells also have a completely alternate means of producing small special-purpose polypeptides which act as antibiotics or various other environmentally-friendly compounds of therapeutic importance. Instead of the ribosome, cells use a very large protein complex called nonribosomal peptide synthetase (NRPS) to produce these compounds. Like a molecular assembly line, the different subunits of an NRPS each perform a critical step in the process and, using moving parts, hand off the product to the next domain. The end product is called a nonribosomal peptide or NRP. Understanding how this process works at the molecular level could lead to new and more powerful therapeutics.

While the chemistry of biological polypeptide synthesis is increasingly well understood, NRPs like antibiotics often contain distinct chemical modifications that are essential to their function. Gramicidin, for example, contains a formyl group at the end of the peptide chain that is clinically important for its antibacterial action. A group from McGill has, for the first time, captured a sequence of structures showing every major step in the special chemical tailoring performed by this NRPS.

Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase, Janice M. Reimer, Martin N. Aloise, Paul M. Harrison & T. Martin Schmeing, Nature (2016). 529, 239-242.

The initial steps in the production of gramacidin happen in three phases: adenylation, thiolation, and formylation. Using crystallography data collected at the Canadian Light Source, Reimer, et al. have captured each phase and found that the NRP is shuttled between domains over a very elongated structure.

Crystallography captures snapshots of molecular structures confined to a crystal lattice. To understand what actually happens in solution, Reimer et al. turned to small angle X-ray solution scattering (SAXS). Using CHESS G1 station’s BioSASXS setup configured for inline size exclusion chromatography (SEC-SAXS), the researchers measured solution scattering profiles for the NRPS modules of interest. Comparison of measured scattering with crystallographic data suggests that the structure is indeed elongated and highly flexible. Modelling of ensembles of possible structures indicates that the biological solution state may consist of a mixture of domains predominately in the formylation state (about 60%) but with a lesser percentage of structures in the thiolation state.

The researchers have produced a short video of the molecular movements that transport the NRP along the elongated complex at each stage of the synthesis.

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Submitted by: Richard Gillilan, MacCHESS, Cornell University