Non-ribosomal peptide synthetase docking domains : structure, function and engineering strategies
Authors:   , Watzel Jonas 
Published: October 2021

Non-ribosomal peptide synthetases (NRPSs) are known for their capability to produce a wide range of natural compounds and some of them possess interesting bioactivities relevant for clinical application like antibiotics, anticancer, and immunosuppressive drugs. The diverse bioactivity of non-ribosomal peptides (NRPs) originates from their structural diversity, which results not only from the incorporation of non-proteinogenic amino acids into the growing peptide chain, but also the formation of heterocycles or further peptide modifications like methylation, hydroxylation and acetylation. The biosynthesis of NRPs is achieved via the orchestrated interplay of distinct catalytic domains, which are grouped to modules that are located on one or more polypeptide chains. Each cycle starts with the selection and activation of a specific amino acid by the adenylation (A) domain, which catalyzes the aminoacyl adenylate formation under ATP consumption. This activated amino acid is then bound via a thioester bond to the 4’-phosphopantetheine cofactor (PPant-arm) of the following thiolation (T) domain. Before substrate loading, the PPant-arm is post-translationally added to the T domain by a phosphopantetheinyl transferase (PPTase), which converts the inactive apo-T domain in its active holo-form. In the last step of the catalytic cycle, two T domain bound peptide building blocks are connected by the condensation (C) domain, resulting in peptide bond formation and transfer of the nascent peptide chain to the following module. Each catalytic cycle is performed by a C-A-T elongation module until the termination module with a C-terminal thioesterase (TE) domain is reached. Here, the peptide product is released by hydrolysis or intramolecular cyclisation. In comparison to single-protein NRPSs, where all modules are encoded on a single polypeptide chain, multi-protein NRPS systems must also maintain a specific module order during the peptide biosynthesis. Therefore, small C-terminal and N-terminal communication-mediating (COM) domains/docking domains (DD) were identified in the C- and N-terminal regions of multi-protein NRPSs. It was shown that these domains mediate specific and selective non-covalent protein-protein interaction, even though DD interactions are generally characterized by low affinities. The first publication of this work focuses on the Peptide-Antimicrobial-Xenorhabdus peptide-producing NRPS called PaxS, which consists of the three proteins PaxA, PaxB and PaxC. Here, in particular the trans DD interface between the C-terminal attached DD of PaxB and N-terminal attached DD of PaxC was structurally investigated and thermodynamically characterized by isothermal titration calorimetry (ITC), yielding a dissociation constant (KD) of ~25 µM, which is a DD typical affinity known from further characterized DD pairs. The artificial linking of the PaxB/C C/NDD pair via a glycine-serine (GS) linker facilitated the structure determination of the DD complex by solution nuclear magnetic resonance (NMR) spectroscopy. In comparison to known docking domain structures, this DD complex assembles in a completely new fold which is characterized by a central α-helix of PaxC NDD wrapped in two V-shaped α-helices of PaxB CDD. The first manuscript of this work focuses on the application of synthetic zippers (SZ) to mimic natural docking domains, enabling the easy assembly of NRPS building blocks encoded on different plasmids in a functional way. Here, the high-affinity interaction of SZs unambiguously defines the order of the synthetases derived from single-protein NRPSs in the engineered NRPS system and allows the recombination in a plug-and-play manner. Notably, the SZ engineering strategy even facilitates the functional assembly of NRPSs derived from Gram-positive and Gram-negative bacteria. Furthermore, the functional incorporation of SZs into NRPS modules is not limited to a specific linker region, so we could introduce them within all native NRPS linker regions (A-T, T-C, C-A). The second publication and the second manuscript of this thesis again focus on the multi-protein PaxS, in particular on the trans interface between the proteins PaxA and PaxB on a molecular level by solution NMR. Therefore, the PaxA CDD adjacent T domain was included into the structural investigation besides the native interaction partner PaxB NDD. Before a three-dimensional structure could be obtained from NMR data, the NH groups located in the peptide bonds had to be assigned to the respective amino acids of the proteins (backbone assignment). Based on these backbone assignments, the secondary structure of PaxA T1-CDD and PaxB NDD in the absence and presence of the respective interaction partner were predicted. The structural and functional characterization of the PaxA T1-CDD:PaxB NDD complex is summarized in manuscript two. The thermodynamic analysis of this complex by ITC determined a KD value of ~250 nM, whereas the discrete DDs did not interact at all. The high-affinity interaction allowed to determine the solution NMR structure of the PaxA T1-CDD:PaxB NDD complex without the covalent linkage of the interaction partners and an extended docking domain interface could be determined. This interface comprises on the one hand α-helix 4 of the PaxA T1 domain together with the α-helical CDD, and on the other hand the PaxB NDD, which is composed of two α-helices separated by a sharp bend. ...