Agroavidin: Extending the Biotin-Based Toolbox

The high affinity biotin based interactions with avidins is exploited as a powerful biotechnological tool for more than 4 decades. The advantages of using tetrameric hen white avidin and bacterial streptavidin as ultra high affinity biotin binders are a proven principle. However, the tetrameric avidins are not suitable for all applications due to their multiple biotin binding sites and unwanted cross-linking. Minimizing the tetrameric assembly was already attempted via protein engineering approaches which led to a drastic decrease in the affinity towards biotin. A different approach was to re-examine and screen sequence databases in order to reveal novel avidin-like proteins that may lead to lower valency high affinity biotin binders. This method led to the discovery of a new subfamily of dimeric avidins that include rhizavidin, hoefavidin and others. Agroavidin, from the agrobactria Rhizobium sp. AAP43, is the most recent member of dimeric avidins that was discovered. Agroavidin has lower affinity towards biotin due to the lack of a conserved Tyr-46 which was already shown to be essential for the high-affinity binding of biotin. In this regard wt-agroavidin did not bind 2-iminobiotin. We have thus mutated Phe-46 into Tyr which resulted in increased binding capabilities towards 2-iminobiotin. In order to clarify the molecular basis of the affinity we resolved the structures of wt and F46Y forms of agroavidin. While the refinement process, we observed to an intriguing feature in the crystal structures that we attempt to develop as a new tool.