Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein–protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold.

Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.

Introduction

The ability to design or identify small molecules that can bind specifically and with high affinity to a given protein is a rate-limiting step in many experiments, including the development of protein microarrays, the analysis of proteins in the context of living cells and the validation of candidate drug targets. In nature, protein–protein interactions can be mediated by small surfaces of folded proteins. This has led to the use of small peptide surfaces presented within the context of a stable protein, called the scaffold, as protein recognition modules. Such reagents, called here peptide aptamers, have been used to disrupt biological protein activity in a range of systems (Table 1). Peptide aptamers are delivered more easily and are more stable in cells than free peptides or antibodies, and their constrained folding results in a low entropic cost of binding and corresponding high affinity for target proteins.¹ The affinity of peptide aptamers for their targets ranges from 10⁻⁶ to 5×10⁻⁹ M,2, 3, 4 compared to K_d 10⁻⁷–10⁻¹¹ M for antibody/target interactions. Nonetheless, peptide aptamers are clearly able to disrupt protein–protein interactions in vivo (see the references in Table 1). Peptide aptamer screens are performed in yeast,⁴ or in mammalian cells,5, 6, 7 which distinguishes them from phage display screens of peptide or antibody libraries performed against potentially misfolded, prokaryotically expressed protein. Protein engineering of peptide aptamers allows them to provide the recognition functionality in the design of a molecular toolkit,⁴ although this potential has yet to be fully realized.

While the most extensively used scaffold is the Escherichia coli protein thioredoxin (TrxA), a number of other proteins have been used, including green fluorescent protein (GFP) (Table 2). The success of this technology hinges upon the robustness of the scaffold, yet one-third of peptides may destabilize GFP (R.W. & P.K.F., unpublished results),⁸ while many TrxA-based peptide aptamers are not expressed stably in cultured human cells (C. Gabernet & P.K.F., unpublished data). Peptides taken out of the context of one scaffold and placed in another frequently lose the ability to interact with their target proteins,⁹ raising the possibility that screens for constrained interactors with a given target may fail unless an appropriate scaffold is used. Finally, the biological activity of most of the scaffolds used to present peptides has not been characterized rigorously, raising the possibility that phenotypes observed when a peptide aptamer is expressed could be due, at least in part, to an effect of the scaffold rather than the inserted peptide. Taken together, these observations indicate the need for further improvements in peptide aptamer technology.

We set out to produce a robust, versatile, biologically neutral scaffold for the presentation of constrained peptides. We sought a protein that could be expressed stably in a range of experimental systems while presenting peptides that are able to interact functionally with a wide range of targets. Such a scaffold would improve peptide aptamer technology substantially by increasing its robustness. In addition, by expanding the repertoire of available scaffolds, our goal is to increase the likelihood that effective hits will be obtained by using libraries in multiple scaffolds in simultaneous screens against each target.

Here, we describe the development of a rigorously tested and biologically inert scaffold for the presentation of constrained peptides, based on human stefin A (SteA), also called cystatin A. SteA is a member of the cystatin family of protein inhibitors of the cathepsin family of cysteine proteases,¹⁰ which are lysosomal peptidases of the papain family. The stefin sub-group of the cystatin family are relatively small (around 100 amino acid residues) single-domain proteins. They receive no known post-translational modification, and lack disulphide bonds, suggesting that they will be able to fold identically in a wide range of extra- and intracellular environments. SteA itself is a monomeric, single-chain, single-domain protein of 98 amino acid residues.11, 12 The structure of SteA has been solved,13, 14, 15 facilitating the rational mutation of SteA into the stefin A triple mutant (STM) scaffold. The only known biological activity of cystatins is the inhibition of cathepsin activity, which allowed us to test exhaustively for residual biological activity of our engineered proteins. Thus, we set out to determine whether protein engineering of native SteA could produce a variant that meets the requirements of an idealised peptide aptamer scaffold. We show that SteA can be engineered to lose its biological activity in vitro and in the cellular context, creating an artificial protein we call STM. Biophysical characterisation shows that the STM scaffold with a peptide inserted retains the folding and thermal stability of the parent protein. We show that STM is able to access both the cytoplasm and the nucleus of human cells, making it a versatile tool for the exploration of the biology of human proteins. The engineered scaffold readily presents peptides for interaction both in vitro and in bacterial, yeast and mammalian cells. Finally, we show that STM is able to present a range of designed peptides that can interact successfully with a known target. We speculate that the peptide aptamer field has been hampered by difficulties in identifying biological activity in cell-based assays, caused, at least in part, by sub-optimal performance of the various existing scaffolds. Our data suggest that we have created a useful scaffold that will be of great benefit to those seeking to study protein–protein interactions in vitro and in vivo.