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bioRxiv · 10.1101/2022.08.03.502643

Design of extended metal-binding β-sandwiches from de novo immunoglobulin domains

Roel-Touris, J.·Nadal, M.·Marcos, E.
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Abstract

The antigen binding sites of antibodies, and derivatives such as scFvs, consist of an immunoglobulin structural framework that anchors hypervariable loops, and that is arranged as a dimer of {beta}-sandwiches (from the heavy and light chains) packing face-to-face. Yet, the naturally occurring dimer orientation of antibodies is not well suited for engineering rigid single-chain formats of increased stability and solubility, which are two key limitations of engineered antibodies. In this work, we computationally designed an extended 12-stranded {beta}-sandwich as a rigid single-chain dimer of de novo immunoglobulin domains interacting through an alternative edge-to-edge arrangement. The extended {beta}-sandwich was found to be hyperstable and structurally accurate as confirmed through X-ray crystallography. We functionalized this design by inserting a long EF-hand calcium-binding motif into the {beta}-hairpin bridge between both domains, showing high metal-binding affinity while remaining soluble and stable in solution. Finally, we propose a few designs with two EF-hand motifs in either or the same side of the scaffold with high-confidence structure predictions; altogether suggesting the robustness and versatility of our scaffold to harbor long functional loops. Our extended {beta}-sandwiches are structurally divergent to natural antibodies and open new avenues for incorporating multiple loop binding sites either for increased or bispecific activities.

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Roel-Touris, J., Nadal, M., Marcos, E.. 2022-08-04. Design of extended metal-binding β-sandwiches from de novo immunoglobulin domains. https://doi.org/10.1101/2022.08.03.502643

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