Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains

Abstract

Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals, but a dynamic domain organization challenges rational design. Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzyme′s buried active site and two binding sites 40 Å apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

Studies of a siderophore-producing cyclization domain: A refined proposal of substrate binding

“…Envisioning extending the C–S bond formed in the cyclization reaction from its length in our docked cyclodehydration intermediate model to a pre-cyclization state reveals the potential for change in this bond-forming coordinate to be accompanied by a large-scale conformational change between open and closed states approximated in our low-frequency normal mode analysis. This is in agreement with the recent solution NMR studies from our coauthor’s laboratory 53 that demonstrate substantial flexibility of the first Cy domain from Ybt synthetase. Katsuyama et al 54 also report disorder in the DXXXXD motif of their Cy structure, therefore flexibility of the two subdomains is likely to be a feature of the entire family.…”

“…Interestingly, protein alignments improve when considering either the N-terminal subdomain (residues 1483 to 1665) or the C-terminal subdomain (residues 1666 to 1910) independently, consistent with movement of these subdomains relative to each other within published NRPS Cy domain structures (Fig. 2B) [51][52][53][54][55] . Specifically, in a Cα alignment of the C-terminal subdomains of HMWP2-Cy2 and EpoB-Cy, helices α2 and α4 of HMWP2-Cy2's N-terminal subdomain are shifted in the direction of the N-terminal subdomain β sheet, and helix α3 of HMWP2-Cy2 is shifted toward the latch strand β11.…”

“…Therefore, in addition to competitive inhibitor development based on intermediate mimics, sites at the PCP binding sites or around the floor loop may enable development of an alternative class of inhibitors that target C domain family members. In particular, the floor loop region has been shown to undergo fluctuations in surface electrostatic potential across the conformational variation of the C domain family 53 despite divergent sequence-level features. Conserved motions’ effects on the electrostatic potential and divergent sequences may allow blocking a key conformational transition in a particular Cy domain with some degree of specificity.…”

“…Eight 1 L LB-Miller cultures supplemented with 50 µg/mL kanamycin were inoculated at 16 °C with 5 mL of an overnight culture and allowed to grow for approximately 72 h. Cells were harvested at 5,000 rpm for 15 min in a JLA 9.1000 Beckman rotor at 4 °C. Cell pellets were immediately resuspended in approximately 40 mL of 50 mM Tris (pH 8.0), 100 mM NaCl, 5 mM β-mercaptoethanol, and 5 mM This phenylalanine is in the region of varying electrostatic potential identified recently by Mishra et al 53 , where varying the electrostatic potential may contribute to flipping toward a condensation-competent state. Mutation of this residue was found to decrease cyclodehydration activity to 31.6% of wild type in PchE 55 and to decrease cyclodehydration activity to ~20% of wild type in BmdB (with a concomitant increase in release of the linear peptide intermediate) 52 , clearly demonstrating its importance in Cy domains.…”

“…1 and S2), and their catalytic mechanisms are only partially understood. Five structures of Cy domains have been reported, involved in biosynthesis of epothilones 51 , bacillamide 52 , yersiniabactin 53 , JBIR-34/35 54 and pyochelin 55 . The first two Cy domain structures identified a structural role for aspartates of the characteristic DXXXXD motif in the crystallized state, which was in keeping with observed roles for residues at these positions in crystallized C domains 34,51,52 .…”