Binder H33 is a small protein binder engineered by ribosome display to bind human interleukin 10. Crystals of binder H33 display severe diffraction anisotropy. A set of data files with correction for diffraction anisotropy based on different local signal-to-noise ratios was prepared. Paired refinement was used to find the optimal anisotropic high-resolution diffraction limit of the data: 3.13-2.47 angstrom. The structure of binder H33 belongs to the 2% of crystal structures with the highest solvent content in the Protein Data Bank.

Human interleukin 24 (IL-24) is a multifunctional cytokine that represents an important target for autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers exciting possibilities in basic research and may have applications in therapy. As a proof-of-concept, we developed a strategy based on recombinant soluble protein variants and genetic code expansion technology to photocontrol the binding between IL-24 and one of its receptors, IL-20R2. Screening of non-canonical ortho-nitrobenzyl-tyrosine (NBY) residues introduced at several positions in both partners was done by a combination of biophysical and cell signaling assays. We identified one position for installing NBY, tyrosine70 of IL-20R2, which results in clear impairment of heterocomplex assembly in the dark. Irradiation with 365-nm light leads to decaging and reconstitutes the native tyrosine of the receptor that can then associate with IL-24. Photocaged IL-20R2 may be useful for the spatiotemporal control of the JAK/STAT phosphorylation cascade.

Human Interleukin 24 (IL-24) is an immunomodulatory cytokine that represents an important target for treatment of autoimmune diseases and cancer. Since the biological functions of IL-24 depend on interactions with its cell membrane receptors, on-demand regulation of the affinity between IL-24 and its cognate partners offers many exciting possibilities in basic research and may have also potential applications in therapy. In particular, control of protein-protein interactions by light has recently found increasing interest. As a proof-of-concept, we developed a strategy based on genetic code expansion technology to photo-control the binding between IL-24 and one of its receptors, IL-20R2. Both these proteins are engineered versions of the native proteins that feature high stability and can be recombinantly expressed in E. coli at adequate yields. Introduction of the photocaged non-canonical amino acid ortho-nitrobenzyl-tyrosine (ONBY) at selected positions of IL-20R2 resulted in severe impairment of heterocomplex assembly as determined by microscale thermophoresis. Irradiation of ONBY-substituted IL-20R2 with UV light (365 nm) reconstitutes the canonical tyrosines and subsequently restores the ability of IL-20R2 to recognize IL-24. We envision that photocontrollable IL-20R2 may be useful for the spatiotemporal regulation of signaling pathways in which IL-24 is involved.

Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.

Raw diffraction images for DoBi scaffold variant J61 based on PIH1D1 N-terminal domain - PDB code 7AVC. Primary publication:P.N. Pham, M. Huličiak, L. Biedermannová, J. Černý, T. Charnavets, G. Fuertes, Š. Herynek, L. Kolářová, P. Kolenko, J. Pavlíček, J. Zahradník, P. Mikulecký, B. Schneider. (2021). Protein Binder (ProBi) as a New Classs of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution. Viruses, 13(2), 190.

Interferon-γ receptor 2 is a cell-surface receptor that is required for interferon-γ signalling and therefore plays a critical immunoregulatory role in innate and adaptive immunity against viral and also bacterial and protozoal infections. A crystal structure of the extracellular part of human interferon-γ receptor 2 (IFNγR2) was solved by molecular replacement at 1.8 Å resolution. Similar to other class 2 receptors, IFNγR2 has two fibronectin type III domains. The characteristic structural features of IFNγR2 are concentrated in its N-terminal domain: an extensive π–cation motif of stacked residues KWRWRH, a NAG–W–NAG sandwich (where NAG stands for N -acetyl-D-glucosamine) and finally a helix formed by residues 78–85, which is unique among class 2 receptors. Mass spectrometry and mutational analyses showed the importance of N-linked glycosylation to the stability of the protein and confirmed the presence of two disulfide bonds. Structure-based bioinformatic analysis revealed independent evolutionary behaviour of both receptor domains and, together with multiple sequence alignment, identified putative binding sites for interferon-γ and receptor 1, the ligands of IFNγR2.