Development of activatable recognition proteins using the bacterial adhesin FimH

Abstract

Recognition molecules, such as antibodies, are widely useful in many applications within bioengineering, or life sciences in general. However, the high cost and variability in antibodies has led to the development of alternative protein scaffolds. While many such alternatives have high binding affinity, a range of specificities, and smaller sizes compared to antibodies, they do not have the advantage of regulated binding, a feature that could be hugely useful in controlled release applications, diagnostic tests, bioseparations, and more. Previously, no antibody or alternative scaffold had been used to develop regulated recognition molecules in an efficient and cost-effective way. This work describes the use of the bacterial adhesin FimH as a protein scaffold for recognition molecules whose binding is regulated. FimH is a two-domain protein in which the binding pocket on one domain is allosterically regulated by the other domain, or parasterically regulated by triggers binding adjacent to the ligand in the pocket. This regulation is based on a major conformational change across the binding domain and binding pocket of FimH, which can switch from a loose to a tight conformation. We show here that this binding pocket contains many permissive positions which, when mutated, are tolerated by the protein and result in a new specificity towards both small molecules (i.e. nickel) and large molecules (i.e. Penta-His monoclonal antibody). Moreover, even after mutating the binding pocket, FimH not only still exists in both conformations, but can be induced to shift between both conformations. This results in the creation of new recognition molecules which are regulated parasterically using mannose, or allosterically using the wedging antibody mab21, when the FimH variant has an affinity that varies with conformation. We then expand the capabilities of FimH as a scaffold by building a diversity library in which two of the binding pocket loops previously identified to carry permissive positions are mutated using NNK codons. The resulting library of 3.32x10^6 codons is characterized and then screened using a biomagnetic selection assay against the model target antibody Penta-His. Two high-affinity clones and one moderate-affinity clone are identified from the library with new specificity towards this antibody. Overall, this work describes how a bacterial adhesin with conformation-dependent binding can be used to generate new recognition molecules which maintain the native protein’s regulatory mechanisms. This has not been done previously with an allosteric scaffold, nor has FimH been utilized in this way before. Ultimately, this paper lays the groundwork for a new, useful tool for generating regulated recognition proteins that meet the ever-changing needs of life sciences research and applications.