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New ways to connect proteins and study their plasticity

Two recent papers from Mark Howarth, Michael Fairhead and colleagues describe how they have exploited one of the most widely used tools in biomedical research.

The papers (1,2) outline work on the streptavidin/biotin system whose versatility stems from the exceptionally tight binding of biotin to streptavidin. As a result, streptavidin can be used for the sensitive detection of biotinylated ligands such as drugs, or for clustering ligands into tetravalent assemblies.

The construction of SpyAvidin tetramer hubs, using a combination of streptavidin/biotin and SpyTag/Catcher, enables the orthogonal assembly of eicosamers (20 subunits)

The construction of SpyAvidin tetramer hubs, using a combination of streptavidin/biotin and SpyTag/Catcher, enables the orthogonal assembly of eicosamers (20 subunits)
(Click to enlarge)

The latter application is currently restricted to assembly of only a single ligand. Attempts to develop a second robust interaction with streptavidin, to increase the potential for nanoassembly applications, have had limited success because the stability of binding was often inferior to that of biotin.

Dr Howarth and colleagues wanted to try to engineer robust orthogonal interaction into the streptavidin/biotin hub by combining it with their own 'superglue' technology, SpyTag/Catcher. SpyTag is a peptide the group developed which forms a spontaneous isopeptide bond to its protein partner SpyCatcher.

They found that they could fuse SpyTag/SpyCatcher with streptavidin/biotin, allowing the formation of chimaeric 'SpyAvidin' tetramers with subunits able to bind biotin, SpyTag or SpyCatcher.

Working with mass spectrometry expert Professor Carol Robinson in the Chemistry Department, they went on to show that when they mixed these tetramers, they could generate assemblies of 8 subunits (octamers) or 20 subunits (eicosamers).

An important application of the streptavidin system is in monitoring specific immune responses. MHC class I molecules, which individually bind very weakly to the T cell receptor, can be clustered using streptavidin. This greatly enhances binding so that MHC class I/streptavidin tetramers can be used to label blood samples and follow what T cell responses are present.

The researchers tested out SpyAvidins to see how well they would perform in such assays. 'We wanted to see whether we could increase the clustering of MHC to T cells using SpyAvidin and see a change in signalling,' explains Dr Howarth.

In collaboration with Professor Anton van der Merwe in the Dunn School of Pathology, they found that the higher order clustering of MHC promoted by SpyAvidin octamers and eicosamers triggered increased stimulation of T cell signalling.

Dr Howarth says that the SpyAvidin-generated assemblies could prove useful when extra sensitivity is required. This might include isolating T cells with particularly low affinity receptors that can arise in autoimmune disease or in response to a tumour.

In the second paper (2), Dr Howarth and colleagues describe a new approach to explore the exceptional strength of the streptavidin/biotin interaction and what happens when a force is applied to it.

'Forces happen naturally on protein interactions and many effects in cell biology, such as sensing faithful chromosome segregation, depend on force,' explains Dr Howarth.

Although it is possible to measure some of the forces involved in these events, or carry out molecular simulation of them, the group wanted to achieve something new - to study experimentally what was happening at atomic resolution.

So they set up a system using streptavidin/biotin as their model in which they gradually introduced repulsive forces chemically into the interaction and trapped and analysed intermediates structurally.

Structures of three Love-Hate ligands in complex with streptavidin. Graphic: Karl Harrison

Structures of three Love-Hate ligands in complex with streptavidin. Graphic: Karl Harrison
(Click to enlarge)

They introduced repulsion, or 'hate', by adding pincer-like arms to biotin to generate a series of conjugates ('Love-Hate ligands'). The modifications were designed to leave the biotin core unchanged but introduce steric clash at positions distant to the binding site.

To synthesise the ligands, they teamed up with Professor Tim Donohoe at OxSynC, an initiative in the Chemistry Department that provides expertise in synthetic chemistry.

With the help of Dr Ed Lowe in the Biochemistry Department, the group crystallised the Love-Hate ligands in complex with streptavidin and determined their structures.

The structures demonstrate the plasticity of the streptavidin/ligand interaction. Parts of the streptavidin are able to flex and its binding contacts are distorted in these complexes. 'We found that most of the biotin ligand stays in its binding site and that part of the streptavidin gets pushed out of the way to accommodate the ligand and counter the 'hate' clashes,' explains Dr Howarth.

The apparent distribution of strain through the protein structure and possibly through the ligand as well suggests that 'love' can override repulsive forces to some extent. Building further repulsion in the system would test how far this could be stretched.

Dr Howarth adds that the group chose a simple system in which to develop the Love-Hate ligand approach, but the approach is one that could potentially be explored in other biological systems where force has an impact on function.


  1. SpyAvidin Hubs Enable Precise and Ultrastable Orthogonal Nanoassembly.
    Fairhead M, Veggiani G, Lever M, Yan J, Mesner D, Robinson CV, Dushek O, van der Merwe PA, Howarth M. J Am Chem Soc. 2014 Aug 21. doi: 10.1021/ja505584f [Epub ahead of print]
  2. Love-Hate ligands for high resolution analysis of strain in ultra-stable protein/small molecule interaction. Fairhead M, Shen D, Chan LK, Lowe ED, Donohoe TJ, Howarth M. Bioorg Med Chem. 2014 Jul 24. doi: 10.1016/j.bmc.2014.07.029. [Epub ahead of print]






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