New Disulphide Bond in Cystatin-Based Protein Scaffold Prevents Domain-Swap-Mediated Oligomerization and Stabilizes the Functionally Active Form.

Matja Zalar,Alexander P Golovanov

doi:10.1021/acsomega.9b02269

Abstract

Peptide aptamers built using engineered scaffolds are a valuable alternative to monoclonal antibodies in many research applications because of their smaller size, versatility, specificity for chosen targets, and ease of production. However, inserting peptides needed for target binding may affect the aptamer structure, in turn compromising its activity. We have shown previously that a stefin A-based protein scaffold with AU1 and Myc peptide insertions (SQT-1C) spontaneously forms dimers and tetramers and that inserted loops mediate this process. In the present study, we show that SQT-1C forms tetramers by self-association of dimers and determine the kinetics of monomer–dimer and dimer–tetramer transitions. Using site-directed mutagenesis, we show that while slow domain swapping defines the rate of dimerization, conserved proline P80 is involved in the tetramerization process. We also demonstrate that the addition of a disulphide bond at the base of the engineered loop prevents domain swapping and dimer formation, also preventing subsequent tetramerization. Formation of SQT-1C oligomers compromises the presentation of inserted peptides for target molecule binding, diminishing aptamer activity; however, the introduction of the disulphide bond locking the monomeric state enables maximum specific aptamer activity, while also increasing its thermal and colloidal stability. We conclude that stabilizing scaffold proteins by adding disulphide bonds at peptide insertion sites might be a useful approach in preventing binding-epitope-driven oligomerization, enabling creation of very stable aptamers with maximum binding activity.

Highlights

Peptide aptamers are proteins that consist of short targetbinding polypeptide loops embedded within a stable protein scaffold, designed to bind to a defined target
ACS Omega shown in the original publication[11] that SQT retains the secondary structure upon various peptide insertions, we have demonstrated in our previous study that an SQT variant, named SQT-1C, with AU1 and Myc peptides inserted into loop 1 and loop 2, respectively, has decreased thermal stability and poor solution behavior.[10]
Slow kinetics and high apparent activation energy of SQT-1C dimerization are consistent with large structural rearrangement needed for dimer formation

Summary

Introduction

Peptide aptamers are proteins that consist of short targetbinding polypeptide loops embedded within a stable protein scaffold, designed to bind to a defined target. Engineered protein scaffolds are typically based on small native globular proteins, modified to remove original function and include new subcloning sites for adding the interchangeable loops. Peptide aptamers are applied in various research tasks, including the development of combinatorial protein libraries for protein recognition,[2,3] studies of protein function and their interactions,[4] diagnostic tools,[5] biosensors,[6] imaging agents,[7] and as biotherapeutics.[8] As such, peptide aptamers are an emerging valuable alternative to monoclonal antibodies which until now have prevailed as the “gold standard” for affinity binding studies

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Results

Conclusion