Elucidating and exploiting docking domain-mediated carrier protein recognition in natural product megasynthetases

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

Bioactive natural products from plants and microorganisms have numerous important applications in medicine and agriculture. They are used to tackle life-threating conditions, such as bacterial and fungal infections, organ transplant rejection and cancer, and as herbicides, insecticides and fungicides that play an essential role in the protection of food crops. Many natural products are assembled by enzymatic "assembly lines", akin to a car production line. Each component of the assembly line must engage in effective communication with the next to ensure the overall process is efficient. Such communication is typically mediated by dedicated "docking domains" attached to the ends of the components, which interact with each other in a specific way. We have been studying the role played by a particular type of docking domain and its interaction partner in the assembly of enacyloxin IIa. This antibiotic is produced by Burkholderia species and has potent activity against Acinetobacter baumannii, a bacterium that causes life threatening diseases in humans for which there is a critical need to find effective new treatments. Our data have shown that this type of docking domain is involved in the assembly of many more bioactive natural products than previously thought, including several anti-cancer agents and an antibiotic that are used in the clinic.
In this project we aim to use a combination of established and recently-developed techniques to develop a better understanding of the way the docking domain involved in the production of enacyloxin recognises its interaction partner. The insights we obtain will be used to modify the enacyloxin assembly line to see whether it behaves in the way we predict. We also aim to investigate a related system in which two components, both of which have a docking domain that is similar to the one involved in enacyloxin production, appear to interact with the same partner to execute sequential tasks in the assembly of the aeruginosins, an unusual group of protein degradation inhibitors produced by cyanobacteria. This will broaden our understanding of the role played by docking domains in natural product assembly and allow us to establish the common principles underlying the way in which they recognise their interaction partners. Finally, we will investigate whether our understanding of these common principles can be harnessed to substitute one of the components of the aeruginosin assembly line with the corresponding component from the enacyloxin system. Overall, this project will significantly deepen our understanding of the roles played by an important type of docking domain in natural product assembly and will establish the feasibility of exploiting such docking domains to construct engineered assembly lines capable of producing novel natural product hybrids.

Technical Summary

Modular polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs) and hybrid PKS-NRPSs typically consist of several subunits that must interact with each other in a programmed manner to ensure a high degree of fidelity in the overall biosynthetic process. Interactions between subunits are typically mediated by various types of structurally complementary N- and C-terminal docking domains (DDs).
This project builds on recent collaborative BBSRC-funded work by the applicants showing that a type of DD found at the N-terminus of amide-forming condensation (C) domains and thiazoline forming heterocyclisation (Cy) domains, and previously thought to be associated with only a handful of systems, is in fact present in more than forty NRPS and hybrid PKS-NRPS assembly lines, including several responsible for the biosynthesis of clinically-approved anticancer agents and antibiotics. Moreover our work has revealed that this type of DD is associated with additional classes of catalytic domain, such as ester-forming C domains, oxazoline-forming Cy domains and flavin-dependent chlorinases. Such DDs interact with a largely unstructured peptide appended to the C-terminus of carrier proteins (CPs) to which the substrates for the downstream catalytic domains are covalently attached.
We have determined the structures of an ester-forming C domain with its N-terminal DD attached and its partner CP containing the C-terminal interacting peptide by X-ray crystallography and NMR spectroscopy, respectively. Here we aim to pursue an interdisciplinary approach to understanding the interaction between these two proteins. We also aim to characterise a system in which a halogenase and an amide-forming C domain, each containing an N-terminal DD, appear to interact with same CP to first chlorinate, then elongate its bound substrate. The utility of our insights for guiding rational pathway engineering will be tested by constructing modified and hybrid assembly lines.

Planned Impact

The development of new techniques for natural product bioengineering is an important cornerstone of industrial biotechnology. For example, there is an urgent need to produce novel derivatives of natural product antibiotics that overcome antimicrobial resistance and this is difficult to achieve using chemical synthesis. Also many herbicides, insecticides and fungicides, which play an essential role in the protection of food crops, are natural products and new derivatives with lower toxicity and greater efficacy are needed to feed the burgeoning global population. Therefore, a number of beneficiaries stand to gain from this research, including the UK-based pharmaceutical and agrochemical companies, the researchers employed by the project and, in the long run, the wider public and health-care in the UK.
The researchers employed by the project will receive top-quality training in the methods and philosophy of highly collaborative and cutting-edge interdisciplinary research at the Chemistry/Biology interface. This will ensure that they are attractive potential employees for UK companies across the pharmaceutical, agrochemical and biotechnology sectors, with an ideal skill set to ensure such companies continue to contribute strongly to the UK economy. The training they receive will also equip them to become leading contributors to the development of a knowledge-based bio-economy, which is predicted to become a strong driver of economic growth in the UK, and indeed across Europe, in the coming decades.
An important part of the work to be carried by the researchers employed on this project is the development and delivery to schoolchildren across the West Midlands region of presentations on the important part played by natural products in industrial biotechnology. This will educate the children about the need to develop new methods for producing natural product analogues and will inspire them to take up the challenge of developing a knowledge-based bio-economy by pursuing a career in science.
New strategies for natural product bioengineering will be an important outcome of this project. This will be of benefit to UK-based pharmaceutical, agrochemical and biotechnology companies actively-engaged in the quest to develop novel natural product-based consumer products, which will ultimately be of benefit to wider society.

Publications

10 25 50
 
Description In this project we have used an interdisciplinary approach to characterise the relationship between structure and function of various steps in biosynthesis of an antibiotic enacyloxin and protease inhibitor, aeruginosin. We have discovered which structural features control reactions resulting in ester or amide bond creation and chlorination in these pathways. We have then used the obtained structural and functional knowledge about these systems to create hybrid systems involving proteins from enacyloxin and aerugiunosin pathways in order to make new molecules that are not traditionally produced by them. This work has resulted in creation of new tools for biosynthetic engineering that should aid in making new useful bioactive molecules, e.g. antibiotics. We were also able to use hybrid systems to make new chlorinated compounds not previously observed in nature.
Exploitation Route Our work described in already published and future publications provides basis for other researchers to use the characterised by us docking domains interfaces to perform biosynthetic engineering on other systems thus facilitating routes to new useful molecules. This could be also potentially of interest to biotechnological industry but would require further optimisation.
Sectors Agriculture, Food and Drink,Chemicals,Education,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description (PANACEA) - A Pan-European Solid-State NMR Infrastructure for Chemistry-Enabling Access
Amount € 4,998,891 (EUR)
Funding ID 101008500 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 08/2021 
End 08/2025
 
Description Enabling new characterisation methods for dynamic systems through the upgrade of 700 MHz solution NMR spectrometer
Amount £799,374 (GBP)
Funding ID BB/W020297/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2022 
End 07/2023
 
Description Illuminating and exploiting programmed O-methylation in trans-AT polyketide synthases
Amount £795,019 (GBP)
Funding ID BB/W003171/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2021 
End 09/2024
 
Description NMR at 1.2 GHz: A World-Leading UK Facility to Deliver Advances in Biology, Chemistry, and Materials Science
Amount £16,836,161 (GBP)
Funding ID EP/X019640/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2023 
End 12/2028
 
Description Renewal of the 600 MHz solid-state NMR console for biological applications
Amount £278,812 (GBP)
Funding ID BB/T018119/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2020 
End 04/2021
 
Description Solid state and solution NMR spectroscopy and cryo-electron microscopy methodology for the characterisation of aggregation mechanisms in proteins
Amount £102,026 (GBP)
Funding ID BB/V50967X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2020 
End 09/2024
 
Description The UK High-Field Solid-State NMR National Research Facility
Amount £2,431,377 (GBP)
Funding ID EP/T015063/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2020 
End 01/2025
 
Title Data relating to the Thesis: Organic carbon across the terrestrial-to-aquatic continuum: Assessing source and delivery processes using a combined fingerprinting and carbon loss modelling approach 
Description Data relating to the Thesis: Organic carbon across the terrestrial-to-aquatic continuum: Assessing source and delivery processes using a combined fingerprinting and carbon loss modelling approach Soil and sediment data from the Loch Davan catchment, Aberdeenshire. Data includes land use specific: n-alkane concentrations n-alkanes compound-specific stable isotopes neutral lipid fatty acids (NLFA) concentrations NLFA compound-specific stable isotopes Carbon, N and bulk stable isotopes 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
URL https://cord.cranfield.ac.uk/articles/dataset/Data_relating_to_the_Thesis_Organic_carbon_across_the_...
 
Description Enacyloxin biosynthetic engineering 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution Phill Stansfeld (University of Warwick), Neil Oldham (University of Nottingham) and Jian Li (Monash University) on enacyloxin biosynthetic engineering, supported by Monash Warwick Alliance Major Research Collaboration in Antimicrobial Resistance. 01/11/21-31/10/24 (£486,530).
Collaborator Contribution Phill Stansfeld (University of Warwick), Neil Oldham (University of Nottingham) and Jian Li (Monash University) on enacyloxin biosynthetic engineering, supported by Monash Warwick Alliance Major Research Collaboration in Antimicrobial Resistance. 01/11/21-31/10/24 (£486,530).
Impact Multidisciplinary collaboration.
Start Year 2020
 
Description Microtubules binding proteins 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed pilot solid-state NMR measurements on protein samples.
Collaborator Contribution Provided samples.
Impact Preliminary measurements.
Start Year 2018
 
Description Molecular modeling of systems involved in biosynthesis of natural products 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution Performed and analysed molecular dynamics simulations of various enzymes involved in natural products biosynthesis.
Collaborator Contribution Experimental data to contrast against the simulations.
Impact https://doi.org/10.1039/D1SC03478B
Start Year 2021
 
Description Protein aggregation 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are developing NMR based methods for characterising protein aggregation.
Collaborator Contribution Exchange of expertise and help with identifying research targets.
Impact Currently only preliminary results obtained.
Start Year 2020
 
Description Role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, 
Organisation Australian National University (ANU)
Country Australia 
Sector Academic/University 
PI Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Collaborator Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Impact Multidispilinary collaboration.
Start Year 2019
 
Description Role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Collaborator Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Impact Multidispilinary collaboration.
Start Year 2019
 
Description Role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Collaborator Contribution Collaboration of Gregory Challis with Gottfried Otting (Australian National University), David Fairlie (University of Queensland) and Max Cryle (Monash University) on role played by BHDD-SLiM interactions in combinatorial biosynthesis of bicyclic depsipeptide histone deacetylase inhibitors, supported by ARC Centre of Excellence for Innovations in Peptide and Protein Science CE200100012. 22/01/21-21/01/28 ($45,508,212 (AUD)).
Impact Multidispilinary collaboration.
Start Year 2019
 
Description Solid-state NMR of membrane proteins 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution We explore applications of fast magic angle spinning solid state NMR to membrane protein, beta-1 adrenergic receptor.
Collaborator Contribution Our collaborator, Daniel Nietlispach, has provided samples.
Impact No outcomes yet. In progress.
Start Year 2022
 
Description Structures of antibiotic-lipid II complexes 
Organisation University of Warwick
Country United Kingdom 
Sector Academic/University 
PI Contribution We have used combination of solution and solid-state NMR (including 100 kHz spinning methodology) to solve structures of antibiotics in complexes with lipid II to inform rational drug development efforts.
Collaborator Contribution Synthesize lipid II.
Impact No outcomes yet. Publication in preparation.
Start Year 2015
 
Description "New Approaches to Drug Discovery and Development: How to move from Bench to Bedside" organized by "Health GRP", University of Warwick 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Attended & actively participated in the interactive workshop event "New Approaches to Drug Discovery and Development: How to move from Bench to Bedside" organized by "Health GRP", University of Warwick comprising the national & international stakeholders in the field of drug discovery for expanding own scientific network & research horizons as well as get better understanding of the research policy framework within UK required to translate the research findings within academia to Industrial R& D set-up.
Year(s) Of Engagement Activity 2019
URL https://warwick.ac.uk/research/priorities/events-events/?start=13052019&view=daily
 
Description Hosting 6th form student from Myton School for work experience placement (18-22 May 2020 - postponed due to covid) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Hosting 6th form student from Myton School for work experience placement (18-22 May 2020). Unfortunately, the activity was postponed due to Covid-19 pandemic.
Year(s) Of Engagement Activity 2020
 
Description Organisation of an Alpine Conference on Magnetic Resonance in Solids 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact About 200 scientists at different stages of their careers participated in this conference leading to exchange of expertise.
Year(s) Of Engagement Activity 2019
URL https://alpine-conference.org/
 
Description Parliament for Researchers Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Attended & actively participated in the interactive workshop event "Parliament for Researchers Workshop" organized by "UK Parliament's Knowledge Exchange unit" comprising primarily the early career researchers or postdoctoral researchers from University of Warwick for understanding of "how academic research is used in the UK Parliament", and also explore the options to identify opportunities to feed the academic research into Parliament's work.
Year(s) Of Engagement Activity 2020
URL https://warwick.ac.uk/fac/sci/chemistry/chemevents/events?calendarItem=8a1785d86d91612c016da5bec4502...
 
Description ubmission (Greg Challis with Munro Passmore and Lona Alkhalaf) of a 1 minute video entitled "Studying Nature's Drug Factories" to Nature Publishing Group's Science in Shorts competition (Feb 2022). 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Submission of a 1 minute video entitled "Studying Nature's Drug Factories" to Nature Publishing Group's Science in Shorts competition.
Year(s) Of Engagement Activity 2022