Developing and investigating an ultra-stable molecular hub for bionanotechnology

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

When biologists wish to isolate or immobilise an object (proteins, DNA, RNA, sugars, small organic molecules, nanoparticles), they will very often attach the vitamin biotin to this object, so that the object can be captured by the protein streptavidin. The link from biotin to streptavidin is one of the strongest known in nature, but this link can be broken in challenging situations, so limiting many biological experiments and devices. We have made an altered streptavidin which forms an even stronger link to biotin, so that biotin is captured for longer and stays bound in harsher conditions. We plan to determine at the atomic level why this new interaction is stronger. This insight should help to illuminate the basis of ultra-strong binding, and so assist the design of drugs that can bind more stably to their targets and thus be more effective. We will also engineer streptavidin so that it can anchor two different kinds of molecules, enabling biologists to create new assemblies to combine and control the functions of biomolecules. We ourselves will use these assemblies to explore how growth factors can either work together or compete in transmitting signals inside the cell, so determining whether the cell lives, dies, or divides.

Technical Summary

The binding of biotin to streptavidin is one of the most widely used interactions in biological research and biotechnology. Streptavidin binds biotin-conjugates with exceptional stability, but dissociation does occur and can be a limitation in imaging, DNA amplification, and nanotechnology. We have identified a mutant streptavidin, which we call traptavidin, showing more than 10-fold slower biotin dissociation, increased mechanical strength, and improved thermostability: this resilience should find diverse applications in biomolecular analysis and assembly. We propose to use crystallography and isothermal titration calorimetry to determine the structural and energetic factors responsible for this tenacious protein-ligand interaction. This understanding should illuminate the intermolecular interactions and dynamics that can confer femtomolar affinity. Streptavidin has 4 biotin-binding sites of equal affinity but we have developed a way to control exactly how many of the biotin binding sites are active, so that harnessing the strength of streptavidin binding does not have to be accompanied by 4-way cross-linking. In this work we will develop tetramers that can stably bind 0-4 copies of two distinct ligands in a precisely defined orientation, so that streptavidin can act as a molecular hub for assembly in synthetic biology and bionanotechnology. We will assemble a hub that precisely orients two different tyrosine kinase receptors, to determine the spatial effects on cross-talk between two signalling pathways important for cell growth, differentiation and survival.

Planned Impact

Who will benefit from this research? Development of orthogonal and single-chain streptavidin tetramers may be beneficial to a range of biotechnology companies, and also to pharmaceutical companies who perform protein-based research. Products developed by these companies could lead to enhanced therapies or diagnostic approaches and so be beneficial to the general public and the National Health Service. How will they benefit from this research? The new streptavidin variants may allow stable, simple and defined-orientation protein immobilisation in biosensors, columns for purification, and protein microarrays. Traptavidin may increase sensitivity in immunoassays and enhance the stability of tracking in microscopy. The new streptavidin variants may directly comprise part of a commercial product/kit, or facilitate research leading to the generation of other products. Understanding the cross-talk from EGFR will help to understand the effects of IGF1R in aging, diabetes and cardiovascular disease and so may contribute to manipulation of IGF1R in medicine, which is already a growing therapeutic target. The likely time-scale is for commercial licensing of these new streptavidin variants is in the second half of 2013. This project will provide important training for the postdoctoral researcher in developing and executing a project which generates new tools and applies them in cell biology. The researcher will be exposed to a wide range of techniques, many of which are likely to be new, including quantitative fluorescent microscopy, crystallography and structure-based protein engineering. The postdoctoral researcher will be supported in the development of their presentation skills and career development by presenting their work within the university and at national and international conferences and by interacting with the media, whenever possible, following successful completion of a milestone. What will be done to ensure that they benefit from this research? Publication in high impact peer-reviewed international journals is the most effective way for us to communicate our findings to potential industrial partners, as well as to fellow academics. Similarly, many of the conferences that the PI attends in chemical biology, proteomics and biophysics contain presenters and audience members from both industry and academia. At this stage we have not engaged with beneficiaries but Isis Innovation are beginning negotiations based on our traptavidin patent. We will follow the same route for protection and dissemination of new streptavidin variants arising from this proposal. Isis Innovation has substantial experience in licensing and links to many UK and international companies but may well establish new links. The PI has previously attended 2 meetings of the Sensors and Instrumentation Knowledge Centre and my laboratory members have presented posters here. The PI will seek to give a talk at a future meeting of this network. As we achieve key results in this project we will communicate with the University of Oxford press office and the BBSRC to ensure that significant findings are communicated widely, to the general public locally and nationally. We will publish detailed protocols to facilitate adoption of the streptavidin variants by other users, as we have done previously for the acceptor peptide and monovalent streptavidin in Nature Protocols. We will also provide rapid e-mail feedback as we have done for the laboratories using biotin ligase and monovalent streptavidin for diverse applications.

Publications

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Dubacheva GV (2017) Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin. in Journal of the American Chemical Society

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Fairhead M (2014) SpyAvidin hubs enable precise and ultrastable orthogonal nanoassembly. in Journal of the American Chemical Society

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Fairhead M (2014) Plug-and-play pairing via defined divalent streptavidins. in Journal of molecular biology

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Fairhead M (2015) Site-specific biotinylation of purified proteins using BirA. in Methods in molecular biology (Clifton, N.J.)

 
Description The first objective of the project to understand structurally the superior stability of traptavidin has been largely fulfilled and the results reported in:

Chivers CE, Koner AL, Lowe ED and Howarth M. (2011) How the biotin-streptavidin interaction was made even stronger: investigation via crystallography and a chimaeric tetramer. Biochemical Journal 435 (1): 55-63.



This paper describes 1.5 Å resolution X-ray structures of our streptavidin mutant, traptavidin. An analysis of the biotin bound (PDB 2Y3F) and free form (PDB 2Y3E) of the traptavidin tetramer shows some interesting structural features. In the published structures of the apo form (biotin-free) of wild-type streptavidin, the loop (L3/4) present near the biotin's valeryl tail is typically highly disordered or in an open conformation. Upon the binding of biotin, this loop closes and becomes highly ordered. Our structural data shows that in both the apo and holo forms of traptavidin the L3/4 loop is closed and ordered. The reduced flexibility of the loop helps to explain the observed slower on-rates and off-rates for biotin found with traptavidin. We also observed that Serine45 in holo traptavidin consistently forms a hydrogen bond to biotin, while this interaction is more erratic in wild-type streptavidin. The reduced breakage of this bond likely inhibits the initiating event in biotin's dissociation pathway. These two structural features give insight into the tighter biotin binding observed with traptavidin.



The structural properties of traptavidin were further analyzed by the generation of a chimaeric tetramer containing one traptavidin subunit and three "Dead" subunits (a mutant which does not bind biotin). The low off-rate of traptavidin was preserved in this chimaeric tetramer, demonstrating that intrasubunit effects are most important for traptavidin's desirable properties. This is an important observation as it simplifies the incorporation of traptavidin and its desirable properties into mixed tetramers of streptavidin (an important aspect of our later work).

For the second objective of the project (to understand thermodynamically the superior stability of traptavidin) we performed differential scanning calorimetry experiments on apo and biotin bound forms of traptavidin, as well as wild type streptavidin as a reference. These experiments show an increased Tm of traptavidin as compared to wild type in both the apo (85.4 vs 75.1°C) and holo forms (116.4 °C vs. 112.4°C), as well as providing some thermodynamic information.



To explicitly define the role of enthalpy and entropy in the biotin-traptavidin interaction, we have performed isothermal titration calorimetry (ITC) experiments. However, due to the extremely high affinity of traptavidin for its ligand, analysis of these experiments was challenging due to the difficulty in collecting data points for partially filled traptavidin, so we used ITC-ligand displacement (where a low affinity ligand is displaced by a higher affinity one) to give better results through collaboration.



In our paper Plug-And-Play Pairing Via Defined Divalent Streptavidins Fairhead M, Krndija D, Lowe ED, Howarth M. Journal of Molecular Biology 2014 Jan 9;426(1):199-214

we generated and obtained the X-ray crystal structures for divalent streptavidin variants with biotin binding subunits in cis or trans. These defined divalent streptavidins (or traptavidins) should be valuable tools in a range of molecular biology and nanotechnology applications. A further manuscript building on these findings, for precise nanoassembly, was published, combining the SpyTag/SpyCatcher and streptavidin approaches, generating a useful hub for synthetic biology and showing its use for modulating immune stimulation.
Exploitation Route Traptavidin and defined divalent streptavidin with high stability binding and defined orientation should be useful to pharmaceutical companies for protein testing and also for assembly of diagnostics, which may in future be used in the National Health Service. Traptavidin has been expressed on a commercial scale by one UK-based company and is being evaluated by different UK and international companies for a number of applications, including in the immobilization of biomolecules for construction of diagnostic devices. If these tests show superior properties for traptavidin, then the university will pursue further licensing agreements. Traptavidin has been licensed already to one diagnostics company. As we demonstate the advantages of controlled assembly using our defined divalent streptavidin variants, we will follow this same approach to enable the wide exploitation of the intellectual property arising.
Sectors Pharmaceuticals and Medical Biotechnology

URL https://pubs.acs.org/doi/abs/10.1021/ja505584f
 
Description The controlled assembly of streptavidin tools and their linkage to SpyTag/SpyCatcher tools was one component contributing to the founding of the spin-out SpyBiotech, using controlled molecular decoration of virus-like particles to accelerate and enhance the assembly of vaccines.
First Year Of Impact 2017
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description BBSRC project grant
Amount £331,618 (GBP)
Funding ID BB/M02122X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2018
 
Title Defined divalent streptavidins 
Description Streptavidin is a central tool in biological research and biotechnology. We developed a version of streptavidin with 2 binding sites (rather than the usual 4), where one could choose to have these binding sites on the same side of the molecule (cis) or on opposite sites of the protein (trans). This should be a valuable tool for stable bridging with precise control over molecular organisation. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Only released in mid-2014. 
URL https://www.ncbi.nlm.nih.gov/pubmed/24056174
 
Title SpyAvidin for irreversible bridging of two distinct biological components 
Description Streptavidin is a very common tool for molecular assembly, but only binds strongly to biotinylated ligands. By linking streptavidin to SpyCatcher, we generated a hub allowing independent and irreversible linkage of two distinct ligands. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact Method only released in mid-2014. 
URL https://www.ncbi.nlm.nih.gov/pubmed/25111182
 
Company Name SpyBiotech 
Description SpyBiotech was founded in March 2017 and employs researchers involved in vaccine development as well as a business development professional. The company is applying technology based on spontaneous isopeptide bond formation towards accelerating vaccine development for human disease. The principal technology is the SpyTag/SpyCatcher peptide/protein interaction but the company is also applying the orthogonal SnoopTag/SnoopCatcher and SnoopLigase systems. Advancement of protein assembly tools during the early stages of this grant has made a contribution to our understanding of evolution of spontaneous isopeptide bond formation. 
Year Established 2017 
Impact There are 6 full time equivalent posts employed at the company. The company is developing new vaccines and is currently doing pre-clinical testing and seeking to move towards clinical trials.
Website http://www.spybiotech.com