Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors

Figures

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  Figure 1. Examples of naturally occurring ankyrin repeat containing proteins. (A) Ankyrin repeat containing protein p100/NF-κB2 (Q00653). Structure of the C-terminal domain (PDB 4OT9, residues 483-756). The ankyrin repeats are colored in pink. (B) Ankyrin repeat containing protein IκBα (P25963). Structure of IκBα in complex with NF-κB (PDB 1NFI, residues 70-282). The ankyrin repeats domains are colored in pink.

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  Figure 2. Ankyrin repeat consensus sequence. Letter refer to amino acids (1 letter code). Residue are numbered according to the UniProt annotation of the ankyrin repeat motif. In black: conserved residue. In blue: non-conserved residue. In orange: semi-conserved residue. X = any amino acid, except C, G, and P. Z = H, N, or Y. * bacterial sequence consensus. ** archea sequence consensus. The amino acids that are structurally important are highlighted in grey [18,21,28,29,32,33,34,35,36,39].

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  Figure 3. Structure of a monoDARPin domain. (A) Library module. The library module is colored in pink, with the conserved TPLH residues in cyan. Residues then can be randomized are indicated in red. (B) Homology model of monovalent DARPin R2 generated by SWISS MODEL (SIB) using the consensus designed ankyrin repeat domain PDB ID 2xee and the sequence of mono DARPin R2 [39]. The N-cap and C-cap are colored in grey.

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  Figure 4. Schematic representation of a ribosome display selection cycle. An mRNA library encoding the proteins of interest without stop codon is translated in vitro (1). After cooling, the translation yields stable ternary complexes of mRNA, ribosomes and nascent polypeptides. These complexes are used for the binding selection on the immobilized target (2). After binding of the polypeptides to the target protein, unbound complexes are washed away (3). The mRNA of the bound complex is eluted by dissociating the ribosomal complex with EDTA (4). A reverse transcription reaction followed by PCR yields the genetic information of the selected clones (5). The amplified genes can then used as input for the next selection round starting with in vitro transcription (6) or cloned intro plasmids for analysis. (adapted from [56]).

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  Figure 5. Schematic representation of ensovibep and structure of monoDARPin R2. (A) Schematic representation of ensovibep. The protein comprises two HSA-binding DARPin domains for in vivo half-life extension and three DARPin domains that bind the RBD of the SARS-CoV-2 spike trimer. The HSA-binding DARPins contain two internal repeats (indicated in yellow) and the RBD binding DARPins three internal repeats (indicated in pink). The N-Cap and C-Cap repeats are represented in grey. The linker sequences are colored in black. (B) Binding surface of monovalent DARPin R2. Homology model of monovalent DARPin R2 generated by SWISS MODEL (SIB) using the consensus designed ankyrin repeat domain PDB ID 2xee and the sequence of DARPin R2 [39]. The library modules are colored in pink. The side chains of the residues interacting with the RBD of the SARS-CoV-2 spike trimer, as defined by cryo-EM analysis are highlighted in green.