Dynamics and mechanistic interpretations of nonribosomal peptide synthetase cyclization domains

Abstract

Ox-/thiazoline groups in nonribosomal peptides are formed by a variant of peptide-forming condensation domains called heterocyclization (Cy) domains and appear in a range of pharmaceutically important natural products and virulence factors. Recent cryo-EM, crystallographic, and NMR studies of Cy domains make it opportune to revisit outstanding questions regarding their molecular mechanisms. This review covers structural and dynamical findings about Cy domains that will inform future bioengineering efforts and our understanding of natural product synthesis.

Keywords: Allostery; Bioengineering; Cyclodehydration; Heterocycle; Pantetheine; Protein–protein interactions.

Figure 1.. Structures of NRPS Cy domains.

A) Crystal structures of Cy domains [12,13,16,17] alongside their products. Heterocycles are colored pink. In BmdB, the N- and C-terminal subdomains are shaded blue and red, respectively. The latch and floor loop are purple and orange, respectively. B) Three conformations of homodimeric PchE modules determined by cryo-EM [15], with the dimer interface marked by a dashed line. ArCPs are represented in surface. C) Catalytic scheme for Cy domains, including currently available structures resembling proposed catalytic states. Atoms for the heterocycle are colored magenta, and atoms of the water leaving group are blue.

Figure 2.. Motions common to Cy domains.

A,B) Structures [,,–18] aligned by strands flanking the floor loop, viewed from the donor site entrance (A) and acceptor site entrance (B). Pink arrows highlight variations between models. MLS = mobile lobe strands, DSE = donor site entrance, ASE = acceptor site entrance.

Figure 3.. Cy-domain motions may participate in substrate binding and allostery.

Comparison of donor site entrances of A) HMWP2-Cy2, B) PchE-Cy1 and C) LgrA-C [13,15,38] highlights twisting of the floor loop, with different floor loop arginine and motif aspartate conformations, as well as different Ppant and thioester positions (C). Electrophile positions are marked by asterisks and floor loops are colored orange. D) Acceptor site entrances of PchE-Cy1, HMWP2-Cy2, EpoB-Cy, FmoA3-Cy2 and BmdB-Cy2 [,,–17]. A semi-conserved phenylalanine in loop 1 is displayed to help emphasize variation in loop 1 structure. E) Docking model of a cyclodehydration intermediate-bound state for HMWP2-Cy2 (acceptor PCP residues and Ppant are in yellow, cartoon for PCP α3 containing the displayed phenylalanine is omitted for clarity) F) Loaded and unloaded holo acceptor CP complexes in PchE highlight differences in pantetheine placement. Acceptor CP and Ppant are displayed as in panel E, and the pantetheine-only model is displayed with transparency.

Figure 4.. Cy structural dynamics modulate transient contacts for function.

A) The dynamic footprint of HMWP2-Cy1 [11]. Carrier proteins from PchE [15] are superimposed to mark donor and acceptor sites. Cy residues exhibiting relaxation dispersion in solution NMR experiments are shown as spheres. B) Substrate funneling at the donor site may be mediated by a network of dynamic residues. The pink arrow labeled “Mobile Lobe” indicates the direction the mobile lobe would shift to open the donor site entrance. The cyclodehydration intermediate (pink sticks) and the PchE product-bound state (light pink sticks) are shown. Yellow dotted lines are polar contacts, including interactions linking the acceptor site (α1, in gray) to the floor loop (orange) and subsequently α4, which may mediate allosteric effects. C) Proposed role of conformational fluctuations in the catalytic cycle. Gray icons resemble observed states shared by C and Cy domains, whereas the purple icon represents an anticipated Cy domain-specific state. Carrier proteins are salmon and yellow orange.