Journal of Molecular Biology
Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers
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 Kd 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technology hinges upon the robustness of the scaffold, yet one-third of peptides may destabilize GFP (R.W. & P.K.F., unpublished results),8 while many TrxA-based peptide aptamers are not expressed stably in cultured human cells (C. Gabernet & P.K.F., unpublished data).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.
Section snippets
Results
Successful peptide aptamer scaffold proteins need to meet the following criteria: the scaffold should be (1) of known structure, allowing an informed choice of the site for peptide insertion or replacement; (2) stable enough to constrain the folding of a broad range of peptides; (3) flexible enough that its folding is not affected by the insertion of a variety of peptides; (4) biologically neutral, i.e. lack interactions with cellular proteins that could contribute a phenotype; and (5) able to
Discussion
Progress in understanding the biology of human cells is hampered by the difficulty of performing genetic experiments in these diploid or sometimes polyploid cells. Alternative approaches to genetic manipulation seek to target mRNA or proteins directly. Antisense or RNA interference (RNAi) technologies knock down the levels of target proteins and hence affect all of their activities, similar to gene knockouts. In contrast, peptide aptamers have the potential to block individual protein–protein
Plasmids and DNA manipulation
pcDNA3SteA, carrying the SteA ORF under the control of the cytomegalovirus promoter was kindly provided by J.P. Waltho (University of Sheffield, UK). pcDNA3.1 HisA and pcDNA3.1 His/Myc B for the construction of His6-tagged proteins in mammalian cells were purchased from Invitrogen (Paisley, UK), pGILDA from Origene (Rockville, MD, USA). pET30a(+), for the expression of His6-tagged proteins in bacteria was purchased from Novagen (Nottingham, UK) and pGFP2-C2 was from Perkin Elmer (Boston, MA,
Acknowledgements
This work was funded by a Grant-in-Aid from the MRC to the Cancer Cell Unit. R.W. was the recipient of an MRC Studentship, S.L. acknowledges the award of a Commonwealth Scholarship, and J.T.H.Y. is grateful for the support of his family. We thank J.P. Waltho and P. Colas for the generous gifts of plasmids, Anasuya Chattopadhyay and Laura Itzhaki for careful reading of the manuscript, and two referees for their constructive comments on an earlier draft of this manuscript.
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