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

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.