A Nuclear Magnetic Resonance Method for Probing Molecular Influences of Substrate Loading in Nonribosomal Peptide Synthetase Carrier Proteins

Goodrich, Andrew C.; Frueh, Dominique P.

doi:10.1021/bi501433r

Cited by 15 publications

(28 citation statements)

References 24 publications

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“…13 C, 15 N apo-ArCP has previously been assigned 29 and its assignment was used as a starting point for assigning holo- and loaded-ArCP resonances.…”

Section: Methodsmentioning

confidence: 99%

“…Lack of success in studying structures of loaded carrier proteins results in part from rapid hydrolysis of the thioester bond, so we recently proposed a means to bypass this limitation for monomeric substrates. 29 Determining if the loaded substrate directly interacts with the protein core of a CP or remains unbound will shape our understanding of the role it plays in directing protein-protein interactions.…”

Section: Introductionmentioning

confidence: 99%

“…We have previously established a method for analysis of ArCP loaded with salicylate in its native thioester form by nuclear magnetic resonance spectroscopy 29 . The method exploits the non-invasive nature of NMR and isotope editing to allow for sustained NMR measurements on loaded-ArCP.…”

Section: Introductionmentioning

confidence: 99%

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Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts

2015

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Nonribosomal peptide synthetases (NRPSs) are microbial enzymes that produce a wealth of important natural products by condensing substrates in an assembly line manner. The proper sequence of substrates is obtained by tethering them to phosphopantetheinyl arms of holo carrier proteins (CPs) via a thioester bond. CPs in holo and substrate-loaded forms visit NRPS catalytic domains in a series of transient interactions. A lack of structural information on substrate-loaded carrier proteins has hindered our understanding of NRPS synthesis. Here, we present the first structure of an NRPS aryl carrier protein loaded with its substrate via a native thioester bond, together with the structure of its holo form. We also present the first quantification of NRPS CP backbone dynamics. Our results indicate that prosthetic moieties in both holo and loaded forms are in contact with the protein core, but they also sample states in which they are disordered and extend in solution. We observe that substrate loading induces a large conformational change in the phosphopantetheinyl arm, thereby modulating surfaces accessible for binding to other domains. Our results are discussed in the context of NRPS domain interactions.

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“…13 C, 15 N apo-ArCP has previously been assigned 29 and its assignment was used as a starting point for assigning holo- and loaded-ArCP resonances.…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts

2015

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“…Analogous carrier proteins from polyketide synthetases can sequester their molecular cargo, although the biochemical relevance of this mechanism is not known 31, 32, 33. In the case of PCPs, the Ppant arm does not appear to interact appreciably with the protein core nor to alter the tertiary structure of the PCP in a significant way 21, 34. This observation supports the “swinging arm hypothesis”, in which the flexible Ppant arm delivers substrates to adjacent domains and the PCP domain serves as a largely rigid and chemically inert platform.…”

Section: The Peptidyl Carrier Proteinmentioning

confidence: 99%

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

et al. 2016

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The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.

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“…However, the natural thioester between the pantetheine and substrate is susceptible to hydrolysis in aqueous solution, and this instability is further aggravated under conditions for NMR studies. 7a,16 Therefore, we prepared pantetheine mimetics with Pro and pyrrole for attachment onto PltL employing an amide linkage in lieu of the thioester. The prolyl- N -pantetheine analog was synthesized by coupling N -Boc-L-Pro to protected pantetheinamine under EDC coupling conditions.…”

mentioning

confidence: 99%

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

Jaremko

Opella

et al. 2015

J. Am. Chem. Soc.

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Type II non-ribosomal 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 biosynthetic regulation in type II NRPSs.

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A Nuclear Magnetic Resonance Method for Probing Molecular Influences of Substrate Loading in Nonribosomal Peptide Synthetase Carrier Proteins

Cited by 15 publications

References 24 publications

Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts

Solution Structure of a Nonribosomal Peptide Synthetase Carrier Protein Loaded with Its Substrate Reveals Transient, Well-Defined Contacts

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

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

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