Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

Mayerthaler, Florian; Feldberg, Anna‐Lena; Alfermann, Jonas; Sun, Xiyan; Steinchen, Wieland; Yang, Han‐Kwang; Mootz, Henning D.

doi:10.1039/d0cb00220h

Cited by 18 publications

(36 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The active site of the PigG–PigI and PigG–PltF structures resembles the active site of the previously reported crosslinked PltL–PltF complex (PDB ID: 6O6E) . Similar to what is observed in the PltL–PltF structure, the A sub domain catalytic lysine responsible for adenylation in PltF and PigI, Lys486 and Lys477, respectively, are 24 and 25 Å away from the active site, in accordance with the proposed domain reorganization process that occurs between the catalysis of the adenylation and thiolation half-reactions. , …”

Section: Resultssupporting

confidence: 85%

“…10 Similar to what is observed in the PltL−PltF structure, the A sub domain catalytic lysine responsible for adenylation in PltF and PigI, Lys486 and Lys477, respectively, are 24 and 25 Å away from the active site, in accordance with the proposed domain reorganization process that occurs between the catalysis of the adenylation and thiolation half-reactions. 11,12 Protein−Protein PCP-A Domain Interface. The protein−protein interface between PigG and both A domains is formed by PigG loop 1, which is a 20-residue region connecting helices 1 and 2 (Figure 2), that mainly contacts the A sub domain.…”

Section: Crystal Structures Of the Pigg−pigi And Pigg−pltfmentioning

confidence: 99%

See 1 more Smart Citation

Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition

et al. 2022

View full text Add to dashboard Cite

Non-ribosomal peptides play a critical role in the clinic as therapeutic agents. To access more chemically diverse therapeutics, non-ribosomal peptide synthetases (NRPSs) have been targeted for engineering through combinatorial biosynthesis; however, this has been met with limited success in part due to the lack of proper protein–protein interactions between non-cognate proteins. Herein, we report our use of chemical biology to enable X-ray crystallography, molecular dynamics (MD) simulations, and biochemical studies to elucidate binding specificities between peptidyl carrier proteins (PCPs) and adenylation (A) domains. Specifically, we determined X-ray crystal structures of a type II PCP crosslinked to its cognate A domain, PigG and PigI, and of PigG crosslinked to a non-cognate PigI homologue, PltF. The crosslinked PCP-A domain structures possess large protein–protein interfaces that predominantly feature hydrophobic interactions, with specific electrostatic interactions that orient the substrate for active site delivery. MD simulations of the PCP-A domain complexes and unbound PCP structures provide a dynamical evaluation of the transient interactions formed at PCP-A domain interfaces, which confirm the previously hypothesized role of a PCP loop as a crucial recognition element. Finally, we demonstrate that the interfacial interactions at the PCP loop 1 region can be modified to control PCP binding specificity through gain-of-function mutations. This work suggests that loop conformational preferences and dynamism account for improved shape complementary in the PCP-A domain interactions. Ultimately, these studies show how crystallographic, biochemical, and computational methods can be used to rationally re-engineer NRPSs for non-cognate interactions.

show abstract

Section: Resultssupporting

confidence: 85%

Section: Crystal Structures Of the Pigg−pigi And Pigg−pltfmentioning

confidence: 99%

Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition

et al. 2022

View full text Add to dashboard Cite

show abstract

“…It is intriguing to speculate that subdomain swapping might have slowed down a conformational change needed to deliver the donor substrate to the C-domain, which is now affected by reversion mutations in the MS and STAP variants. 47 Strikingly, it follows from our two-step model of NRPS specificity that A-domain dominates C-domain specificity, which is illustrated by simulations of a hypothetical twomodule system with tailored acylation and condensation constants (Figure 5). The simulations show that C-domain rate constants matter for the rate of product formation, but not for the specificity.…”

Section: Discussionmentioning

confidence: 76%

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Stanišić

Hüsken

Stephan

et al. 2021

Preprint

View full text Add to dashboard Cite

<div> <p>Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A- and C-domains determines the product specificity of NRPSs.</p> </div>

show abstract

“…It is intriguing to speculate that subdomain swapping might have slowed down a conformational change needed to deliver the donor substrate to the Cdomain, which is now affected by reversion mutations in the MS and STAP variants. 50 Strikingly, it follows from our two-step model of NRPS specificity that A-domain in many cases overrides C-domain specificity, which is illustrated by simulations of a hypothetical two-module system with tailored acylation and condensation constants (Figure 5). The show that C-domain rate constants matter for the rate of product formation but not for specificity.…”

Section: ■ Discussionmentioning

confidence: 78%

“…These differences might indicate an influence of A-domain mutations on a reaction step after T-domain acylation. It is intriguing to speculate that subdomain swapping might have slowed down a conformational change needed to deliver the donor substrate to the C-domain, which is now affected by reversion mutations in the MS and STAP variants …”

Section: Discussionmentioning

confidence: 99%

Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

et al. 2021

View full text Add to dashboard Cite

Engineering of nonribosomal peptide synthetases (NRPS) has faced numerous obstacles despite being an attractive path towards novel bioactive molecules. Specificity filters in the nonribosomal peptide assembly line determine engineering success, but the relative contribution of adenylation (A-) and condensation (C-)domains is under debate. In the engineered, bimodular NRPS sdV-GrsA/GrsB1, the first module is a subdomain-swapped chimera showing substrate promiscuity. On sdV-GrsA and evolved mutants, we have employed kinetic modelling to investigate product specificity under substrate competition. Our model contains one step, in which the A-domain acylates the thiolation (T-)domain, and one condensation step deacylating the T-domain. The simplified model agrees well with experimentally determined acylation preferences and shows that the condensation specificity is mismatched with the engineered acylation specificity. Our model predicts changing product specificity in the course of the reaction due to dynamic T-domain loading, and that A-domain overrules C-domain specificity when T-domain loading reaches a steady-state. Thus, we have established a tool for investigating poorly accessible C-domain specificity through nonlinear kinetic modeling and gained critical insights how the interplay of A-and C-domains determines the product specificity of NRPSs.File list (2) download file view on ChemRxiv Stanisic_manuscript_sdVGrsA .pdf (1.29 MiB) download file view on ChemRxiv Stanisic_Supplementary_Information_sdVGrsA.pdf (3.04 MiB)

show abstract

Intermediary conformations linked to the directionality of the aminoacylation pathway of nonribosomal peptide synthetases

Abstract: In-solution analysis of conformational changes of NRPS adenylation and peptidyl-carrier protein domains under catalytic conditions reveals a new intermediary conformation.

Cited by 18 publications

References 52 publications

Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition

Essential Role of Loop Dynamics in Type II NRPS Biomolecular Recognition

An Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Engineered Nonribosomal Peptide Synthetase Shows Opposite Amino Acid Loading and Condensation Specificity

Contact Info

Product

Resources

About