Computational Modeling of Designed Ankyrin Repeat Protein Complexes with Their Targets

Abstract

Recombinant therapeutic proteins are playing an ever-increasing role in the clinic. Highaffinity binding candidates can be produced in a high-throughput manner through the process of selection and evolution from large libraries, but the structures of the complexes with target protein can only be determined for a small number of them in a costly, low-throughput manner, typically by x-ray crystallography. Reliable modeling of complexes would greatly help to understand their mode of action, and improve them by further engineering, e.g., by designing bi-paratopic binders. Designed Ankyrin Repeat Proteins (DARPins) are one such class of antibody mimetics that have proven useful in the clinic, in diagnostics and research. Here we have developed a standardized procedure to model DARPin-target complexes that can be used to predict the structures of unknown complexes. It requires only the sequence of a DARPin and a structure of the unbound target. The procedure includes homology modeling of the DARPin, modeling of the flexible parts of a target, rigid body docking to ensembles of the target and docking with a partially flexible backbone. For a set of diverse DARPin-target complexes tested it generated a single model of the complex that well approximates the native state of the complex. We provide a protocol that can be used in a semi-automated way, and with tools that are freely available. The presented concepts should help to accelerate the development of novel bio-therapeutics for scaffolds with similar properties.

The production of the first functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology

“…Pertuzumab binds to subdomain II in the extracellular domain of HER2, allowing it to bind in the presence of DARPin G3. Results of macromolecular docking on chloroplast-made DARPin G3 with HER2 complex with trastuzumab fab are in agreement with structural modeling and experimental observations that trastuzumab and DARPin G3 binding to HER2 are not competitive with each other [ 61 , 75 , 76 ].…”

Monomer‐scale design of functional protein polymers using consensus repeat sequences

“…Recent advances in computation may further accelerate the discovery and broader application of DARPins through predictive modeling of DARPin-target complexes. 229 An underexplored opportunity for ankyrin protein engineering lies in the conserved residues, such that alterations to conserved regions may change the curvature of ankyrin repeat proteins. Ankyrin scaffolds with sequence-defined curvature may expand the already broad range of target binding partners.…”

An ankyrin repeat chaperone targets toxic oligomers during amyloidogenesis

“…This has motivated the development of small molecules and therapeutic antibodies that target the Aβ peptide and its fibrillation pathway (Ebrahimi‐Fakhari et al, 2011; Ebrahimi‐Fakhari et al, 2013; Linse et al, 2020; Olanow, 2009; Olanow et al, 2008). Besides antibodies, an important group of alternative scaffolds amenable to protein engineering is the ankyrin repeat domain (ARD), a commonly occurring repeat protein scaffold involved in diverse protein–protein interactions (Barrick et al, 2008; Binz et al, 2003; Forrer et al, 2003; Mittl et al, 2020; Radom et al, 2019; Schilling et al, 2022). ARDs comprise multiple repeats of helix‐turn‐helix motifs (AR motifs), which together form a concave and elongated surface for biomolecular recognition.…”