Investigation of protein-ligand and protein-protein interactions in type II non-ribosomal peptide synthetases

Non-ribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of many pharmaceutically relavant compounds. Type II NRPSs are an emerging subfamily of NRPSs that form hybrid pathways with type I fatty acid synthases (FAS), polyketide synthases (PKS), type I NRPSs, or others. The type II NRPSs commonly contain tailoring enzymes that generate unique substrate modifications, such as dehydrogenations and halogenation. Unlike type I NRPSs, the type II systems consists of standalone enzymes, an ideal feature for combintarial biosynthesis and metabolic engineering. Unfortunately, engineering efforts have been met with limited success due to lack of understanding of protein-protein interactions inherent to these pathways.My dissertation work focuses on using structural biology to investigate type II pyrrole containing natural product pathways, specifically, the antifungal agent pyoluteorin and two prodiginine antitumor agents prodigiosin and undecylprodigiosin. Important to pyrrole formation are the peptidyl carrier protein (PCP) and the adenylation (A) domain. The PCP is post-translationally modified by a 4’-phosphopanetetheine group (holo-PCP) at a conserved serine residue, and the terminal thiol serves as the point of attachment for all NRPS intermediates. The A domain facilitates covalent attachment of a specific amino acid to the holo-PCP. The PCP then shuttles the cargo from one tailoring enzyme to the next in an organized fashion (Fig. 1) The two proteins are vital to precursor incorporation into pathways and substrate alteration. In many pathways (including pyoluteorin), a FADH2-dependent halogenase introduces chlorines to the pyrrole. Halogenation is essential for the biological activity of many natural products. Structural and chemo-enzymatic investigation of these three enzymes will aid in future engineering efforts in NRPS pathways. In FAS and PKS pathways, the acyl carrier protein sequesters tethered substrates in a hydrophobic cleft between helix II and III for protection from undesirable reactions. Substrate sequestration in NRPS PCPs has not been demonstrated. To investigate the phenomena, we determined solution NMR structures of the type II PCP PltL, the peptidyl carrier protein from the pyoluteorin pathway (Fig. 2). Naturally, PCP and substrate are covalently attached through a thioester bond, a labile bond known to hydrolyze in aqueous environments. Chemoenzymatic methods were used to stabilize the pyrrolyl-PltL intermediate for protein NMR studies. The structures of both the holo-PltL and pyrrolyl-PltL intermediates were determined as the first functionally characterized type II PCP. The recognition between PCP and A domain is specific in NRPSs. In fact, the homologous pairs from pyoluteorin and undecylprodigiosin pathways are only active with the cognate partner. We analyzed the homologous PCP and A domain from the prodigiosin pathway and, surprisingly, the PCP PigG was a promisicuous substrate for A domains from all three pathways. We decided to structurally investigate the specificity differences between the pyoluteorin PltL and prodigiosin PigG. The solution NMR structure of holo-PigG was determined and compared to the structure of holo-PltL. The structural features of the two proteins are similar, as expected due to the distinct pyrrole PCP family. Although, dynamic simulations revealed significantly more flexibility in holo-PigG. NMR titration experiments revealed the loop 1 region of both PCPs that was significantly perturbed when the A domain partners were introduced (Fig. 4). Mutations to the loop 1 region of PltL and PigG significantly altered the loading activity of the A domains compared to mutations in other regions. The mutant studies further confirmed the importance of loop 1 in PCPs

Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL.

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems

Structure and Substrate Sequestration in the Pyoluteorin Type II Peptidyl Carrier Protein PltL.

Type II nonribosomal peptide synthetases (NRPS) generate exotic amino acid derivatives that, combined with additional pathways, form many bioactive natural products. One family of type II NRPSs produce pyrrole moieties, which commonly arise from proline oxidation while tethered to a conserved, type II peptidyl carrier protein (PCP), as exemplified by PltL in the biosynthesis of pyoluteorin. We sought to understand the structural role of pyrrole PCPs in substrate and protein interactions through the study of pyrrole analogs tethered to PltL. Solution-phase NMR structural analysis revealed key interactions in residues of helix II and III with a bound pyrrole moiety. Conservation of these residues among PCPs in other pyrrole containing pathways suggests a conserved mechanism for formation, modification, and incorporation of pyrrole moieties. Further NOE analysis provided a unique pyrrole binding motif, offering accurate substrate positioning within the cleft between helices II and III. The overall structure resembles other PCPs but contains a unique conformation for helix III. This provides evidence of sequestration by the PCP of aromatic pyrrole substrates, illustrating the importance of substrate protection and regulation in type II NRPS systems

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces

Dynamic visualization of type II peptidyl carrier protein recognition in pyoluteorin biosynthesis.

Using a covalent chemical probe and X-ray crystallography coupled to nuclear magnetic resonance data, we elucidated the dynamic molecular basis of protein recognition between the carrier protein and adenylation domain in pyoluteorin biosynthesis. These findings reveal a unique binding mode, which contrasts previously solved carrier protein and partner protein interfaces

Manipulating Protein–Protein Interactions in Nonribosomal Peptide Synthetase Type II Peptidyl Carrier Proteins

In an effort to elucidate and engineer interactions in type II nonribosomal peptide synthetases, we analyzed biomolecular recognition between the essential peptidyl carrier proteins and adenylation domains using nuclear magnetic resonance (NMR) spectroscopy, molecular dynamics, and mutational studies. Three peptidyl carrier proteins, PigG, PltL, and RedO, in addition to their cognate adenylation domains, PigI, PltF, and RedM, were investigated for their cross-species activity. Of the three peptidyl carrier proteins, only PigG showed substantial cross-pathway activity. Characterization of the novel NMR solution structure of holo-PigG and molecular dynamics simulations of holo-PltL and holo-PigG revealed differences in structures and dynamics of these carrier proteins. NMR titration experiments revealed perturbations of the chemical shifts of the loop 1 residues of these peptidyl carrier proteins upon their interaction with the adenylation domain. These experiments revealed a key region for the protein–protein interaction. Mutational studies supported the role of loop 1 in molecular recognition, as mutations to this region of the peptidyl carrier proteins significantly modulated their activities

Modeling linear and cyclic PKS intermediates through atom replacement.

The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs

Antitumor Activity of 1,18-Octadecanedioic Acid-Paclitaxel Complexed with Human Serum Albumin.

We describe the design, synthesis, and antitumor activity of an 18 carbon α,ω-dicarboxylic acid monoconjugated via an ester linkage to paclitaxel (PTX). This 1,18-octadecanedioic acid-PTX (ODDA-PTX) prodrug readily forms a noncovalent complex with human serum albumin (HSA). Preservation of the terminal carboxylic acid moiety on ODDA-PTX enables binding to HSA in the same manner as native long-chain fatty acids (LCFAs), within hydrophobic pockets, maintaining favorable electrostatic contacts between the ω-carboxylate of ODDA-PTX and positively charged amino acid residues of the protein. This carrier strategy for small molecule drugs is based on naturally evolved interactions between LCFAs and HSA, demonstrated here for PTX. ODDA-PTX shows differentiated pharmacokinetics, higher maximum tolerated doses and increased efficacy in vivo in multiple subcutaneous murine xenograft models of human cancer, as compared to two FDA-approved clinical formulations, Cremophor EL-formulated paclitaxel (crPTX) and Abraxane (nanoparticle albumin-bound (nab)-paclitaxel)

Modeling Linear and Cyclic PKS Intermediates through Atom Replacement

[Image: see text] The mechanistic details of many polyketide synthases (PKSs) remain elusive due to the instability of transient intermediates that are not accessible via conventional methods. Here we report an atom replacement strategy that enables the rapid preparation of polyketone surrogates by selective atom replacement, thereby providing key substrate mimetics for detailed mechanistic evaluations. Polyketone mimetics are positioned on the actinorhodin acyl carrier protein (actACP) to probe the underpinnings of substrate association upon nascent chain elongation and processivity. Protein NMR is used to visualize substrate interaction with the actACP, where a tetraketide substrate is shown not to bind within the protein, while heptaketide and octaketide substrates show strong association between helix II and IV. To examine the later cyclization stages, we extended this strategy to prepare stabilized cyclic intermediates and evaluate their binding by the actACP. Elongated monocyclic mimics show much longer residence time within actACP than shortened analogs. Taken together, these observations suggest ACP-substrate association occurs both before and after ketoreductase action upon the fully elongated polyketone, indicating a key role played by the ACP within PKS timing and processivity. These atom replacement mimetics offer new tools to study protein and substrate interactions and are applicable to a wide variety of PKSs