The multimodular assembly lines of the macrocyclic antibiotics Tyrocidine A, Surfactin A and Gramicidin S and the synthetases of the siderophores vibriobactin and enterobactin are shown in a graphical presentation beside the chemical structure of each assembly line. The assembly lines are represented in respective modules and domains for the synthesis of one full length natural product. The iteratively working Gramicidin assembly line is shown in two copies of its assembly line (black/gray). The chemical structure of Gramicidin S reflects this The definitions of domains: A = Adenylation, C = Condensation, T = Thiolation- or peptidyl carrier protein (PCP) are given in a figure legend. The filled domains within these assembly lines have their structures determined by X-ray crystallography or NMR spectroscopy.

(a) the three conformational states observed for TycC3-PCP in NMR spectra of non-modified apo-PCP (A and A/H-states) and 4′-PP cofactor modified holo-PCP (A/H and H-states); The structures of all three states are shown with numbered helices; (*) marks the position of the active site serine for the post-translational modification. The chemical exchange between two conformers resulted in sets of two signals per amino acid residue in 15N-HSQC spectra. Examples of these double peaks are shown for apo-PCP (A-state) and holo-PCP (A/H-state). Substrate modification of the free HS-4′-PP cofactor arrests the structural dynamic and superposition of slow exchange-typical double peaks from apo- and holo-PCP and single peaks for acetyl-S-4′-PP-holo-PCP are shown (15N-HSQC of F69 and V67 for the H-state). HS marks the position of the 4′-PP thiol function in the A/H- and H-states and demonstrates its displacement on the surface of holo-TycC3-PCP. (b) The exchange ratio of apo-PCP is shifted toward the A-state in interaction with Sfp, while the editing TEII selects misacylated holo-PCP arrested in the H-state. The line-shapes analysis demonstrates the selection of the minor conformation. The diagram (below) shows the allosteric exchange model, dependent on the modification with the 4′-PP cofactor. (c, d) Comparison of the structural complexes of apo-TycC3-PCP : Sfp and ACP : AcpS. The interface of the protein complexes is shown by a mesh surface; illustrations demonstrate the assembly of the proteins in the complexes and the enzymatic function of the PPTases.

(a) Structure of the EntF PCP-TE didomain; (*) indicates position of the active site serine of the PCP. (b) Opening the interface by a rotation along the dashed line allows identification of residues involved in domain-domain recognition. The active site residues of the TE (S180, D207, and H313) are indicated. (c) The TycC3-PCP : SrfTEII complex demonstrates a more compact interface compared to the EntF PCP-TE didomain (a). The inset interface of PCP and TEII shows the position of the modelled 4′-PP thiol relative to the active site residues of the TEII. Illustrations demonstrate the orientation of the domains and the enzymatic functions of the EntF PCP-TE didomain and the PCP : TEII complex.

The PCP domain [(*) indicates the active site serine] faces the opposite direction to the active site histidine (H224) of the Tyc C10 domain. Illustrations indicate the orientation of domains and the enzymatic function of the PCP-C didomain.

(a) The structure of the C-A-PCP-TE module shows the peptidyl carrier domain oriented towards the C domain. The positions of the active site residues of the C domain (His 147), of PCP (*), and of the TE domain (Ser 1120, Asp 1147, His 1247) are indicated. A substrate leucine is bound inside the A domain (#). The interaction surfaces of the peptidyl carrier domain with surrounding domains are shown by mesh surfaces. Illustrations indicate the orientation of domains of the termination module and the enzymatic function of SrfA-C. (b) Rotating the structure reveals a large stable interface between the C and A domains (joint surface). This rigid interface defines the module C-A-PCP as a stable unit in NRPS assembly lines. The positions of the adjacent domains are indicated.