Characterization of the protein ligand binding site on protein disulphide-isomerase (PDI) and its homologues

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

This application is part of a joint proposal with Prof. Robert Freedman at the University of Warwick. Warwick is the lead Research Organisation and the Joint Reference code is D189108. Please refer to the lead application for the Summary.

Technical Summary

We aim to define in detail the interaction between protein disulphide-isomerase (PDI) and its protein substrates, in order to picture its mechanism, illuminate its cellular function and inform work on PDI homologues. We will determine the structure of the b' domain of human PDI (the principal ligand binding domain) in various contexts. Our NMR data on PDI constructs including the b' domain indicate conformational heterogeneity within the b' domain that explains previous failures to solve the structure by x-ray and NMR approaches. Our targets will be constructs whose spectra indicate a single defined structure; the b-b'-x construct comprising all the non-catalytic domains of PDI, and mutants of the wild-type b'-x sequence. We will express them in E.coli and purify 15N or 15N/13C or 15N/13C/2H proteins for high resolution heteronuclear NMR studies. We will assign resonances, generate distance and angle constraints and calculate structures using conventional multi-dimensional NMR approaches. We will explore the dynamic properties of the proteins under study, using relaxation and H/D exchange methods, obtaining site-specific data where possible. We will determine the effects of small unstructured peptide ligands on NMR parameters of these proteins in order to define binding sites and impacts of ligand binding on PDI domain dynamics. We will initiate experiments using incompletely-folded protein ligands, focussed on the structural and dynamic properties of the ligand protein and how these reflect binding to PDI. For this work we will express in E.coli mutants of bovine pancreatic trypsin inhibitor (BPTI) which contain Cys-to-Ser mutations to constrain the disulphides that can be formed. These mutants mimic incompletely folded intermediates on the BPTI disulphide-linked folding pathway. We will use 15N relaxation measurements and transferred NOE approaches to explore the structures of these species when bound non- covalently to PDI and the nature of the interaction.
 
Description We have carried out structural studies on a key 'folding helper' or chaperone, PDI (protein disulphide isomerase). PDI is a crucial component of the machinery that enables secreted proteins (such as antibodies, hormones and growth factors, blood-clotting proteins and digestive enzymes) to fold into their correct shape prior to being secreted from the cell. All these proteins contain a specific cross-link (the disulphide bond) which stabilizes their structure and a crucial aspect of their folding is the rapid formation of the correct set of disulphide cross-links. This process is catalysed and facilitated by PDI and the aim of our project was to understand in molecular detail how PDI does this.

We previously identified one region of PDI (the b' domain) as the region that binds partly-folded proteins (its 'substrates'), and in this project we extended our understanding by:
i) determining the structure of this b' domain in atomic detail ('solving the structure'),
ii) establishing which surface of the b' domain was directly involved in binding to its target partly folded molecules ('mapping the substrate binding site'), and
iii) showing that a neighbouring region of PDI known as the x-linker, can also bind to the binding site on b', and that this provides a removable 'cap' over the binding site so that it is not usually exposed; a substrate molecule can displace the 'cap' and thus gain access to the binding site. This is a key element in understanding how PDI works.

We have also begun to study how PDI binds its substrates in even greater detail. We have shown differences in how unfolded, partly-folded and folded proteins interact with PDI and we have studied how PDI associates with partner proteins in the cell. We have studied the dynamics of PDI to show that the binding-site has intrinsic flexibility, not found in other parts of the molecule, which may enable the site to adapt itself to 'fit' many different partly-folded protein substrates. We have also probed the overall mobility and flexibility of PDI, showing that this reflects the movement of part of the x-linker into and out of the binding site on b'.

We have also started to study the structure and activities of other cellular proteins closely related to PDI.
Exploitation Route The work was primarily directed towards enhancing fundamental knowledge. The primary users of the findings will therefore be other researchers. PDI is widely cited throughout the biotechnological, biomedical and agri-food literature, hence the findings of this work are of interest to a wide range of scientists.

The commercial value of recombinant disulphide bonded proteins is now very significant. There is a substantial market in protein therapeutics and the great majority of these agents are disulphide-bonded human proteins. Recombinant products can be generated in a wide range of systems (E.coli, yeast and other fungi, insect cells and mammalian cells), but the yield of correctly folded and correctly-disulphide-bonded product is a critical issue in all. It is in this area of recombinant protein production that we expect our findings to have the greatest impact.

An important finding in our work was the 'capped' and 'uncapped' conformers of the b' domain of PDI. This reversible 'capping' of the primary substrate binding has the potential to play a key regulatory role in the function of PDI. These findings will be used in future work that looks at the dynamics of PDI interactions with substrates and partner proteins to gain a more complete understanding of how PDI operates as a molecular machine.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
 
Description We are not aware of any direct use of our findings yet beyond academia, however, it may take many years for the full value of our work to materialise. The need to produce recombinant correctly-folded disulphide-bonded proteins is significant and PDI remains a target of immense interest for further improving the host's capacity to produce such proteins in high yield and high fidelity. Our work contributes to a large and active field that will eventually identify methods by which cells can be 'tuned' by engineering for the expressions needs of a particular target protein. The cellular machinery for protein folding is large and complex and a significant amount of investment has been made by industry and government to better understand the process. The ability to increase the yield of recombinant protein production will ultimately benefit the economic competitiveness of the UK.
First Year Of Impact 2009
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic
 
Description Project Grant (joint application with Prof Freedman, University of Warwick)
Amount £239,648 (GBP)
Funding ID 093125/B/10/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2011 
End 07/2014
 
Title Chemical shift assignments for b'x 
Description NMR chemical shift data (1H, 15N, 13C) for the b'x domain of human PDI (BMRB entry 15998) 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Data freely available in the Biological Magnetic Resonance Data Bank. 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=15998
 
Title Chemical shift assignments for bb'x 
Description NMR chemical shift data (1N, 15N, 13C) for the bb'x domains of human PDI (BMRB entry 15974) 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Data freely available in Biological Magnetic Resonance Data Bank 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=15974
 
Title Chemical shift assignments for oxidised ERp18 
Description NMR chemical shift data (1H, 15N, 13C) for human oxidised ERp18 (BMRB entry 15964) 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Data freely available in the Biological Magnetic Resonance Data Bank 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=15964
 
Title Chemical shift assignments for reduced ERp18 
Description NMR chemical shift data (1H, 15N, 13C) for reduced human ERp18 (BMRB entry 7430) 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Data freely available in the Biological Magnetic Resonance Data Bank 
URL http://www.bmrb.wisc.edu/data_library/summary/index.php?bmrbId=7430
 
Title Crystal structure of b'x 
Description Crystal structure of the b'x domain from human PDI (mutant I272A). pdb entry 3BJ5 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact Structure freely available in the Protein Data Bank. Structure reveals 'capping' of the substrate binding site by the x-linker. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=3BJ5
 
Title NMR structure of ERp27 
Description NMR solution structure of the b domain of ERp27 (pdb entry 2L4C) 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? Yes  
Impact Structure freely available in the Protein Data Bank 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=2L4C
 
Title NMR structure of oxidised ERp18 
Description NMR solution structure of oxidised human ERp18 (pdb code 2K8V) 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? Yes  
Impact Data freely available to all in the Protein Date Bank (PDB) 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=2k8v
 
Description LWR 
Organisation University of Oulu
Department Biocenter Oulu
Country Finland 
Sector Academic/University 
PI Contribution Sharing of ideas and expertise.
Collaborator Contribution Sharing of materials, ideas and expertise.
Impact The collaboration contributed to all areas of the work and resulted in the output of several joint publications.
Start Year 2006
 
Description Robert Freedman 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This BBSRC award was joint between Prof R B Freedman at the University of Warwick and myself (Dr R A Williamson, University of Kent). Prof Freedman was Lead PI on the joint research project. The grant number for the joint project at Warwick was BB/D017807/1 Contact between the two teams at Kent and Warwick was frequent and included the exchange of materials, ideas and expertise. All outputs were joint and included contributions from both teams.
Collaborator Contribution Contact between the two teams at Warwick and Kent was frequent and included the exchange of materials, ideas and expertise. All outputs were joint and included contributions from both teams.
Impact All outputs from this work were joint and included contributions from both teams.
Start Year 2006
 
Description Cafe Scientifique Hull 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Lively discussion generated by presentation both immediately after the talk and continuing into the evening in small groups.

Part of a successful and on-going series of presentations
Year(s) Of Engagement Activity 2013