Flexible regions govern promiscuous binding of IL‐24 to receptors IL‐20R1 and IL‐22R1

Abstract

KeywordsIL-24; interleukin 24; promiscuity of receptor binding; protein design; protein stability Correspondence B. Schneider, Ji r ı Zahradn ık and Lucie Kol a rov a have contributed equally to the work.Interleukin 24 (IL-24) is a cytokine with the potential to be an effective treatment for autoimmune diseases and cancer. However, its instability and difficulties in its production have hampered detailed biological and biophysical studies. We approached the challenges of IL-24 production by using the PROSS algorithm to design more stable variants of IL-24. We used homology models built from the sequences and known structures of IL-20 and IL-19 and predicted and produced several extensively mutated IL-24 variants that were highly stable and produced in large yields; one of them was crystallized (IL-24B, PDB ID 6GG1; 3D Interactive at http://pro teopedia.org/w/Journal: FEBS_Journal:1). The mutated variants, however, lost most of their binding capacity to the extracellular parts of cognate receptors. While the affinity to the receptor 2 (IL-20R2) was preserved, the variants lost affinity to IL-20R1 and IL-22R1 (shared receptors 1). Back engineering of the variants revealed that reintroduction of a single IL-24 wild-type residue (T198) to the patch interacting with receptors 1 restored 80% of the binding affinity and signaling capacity, accompanied by an acceptable drop in the protein stability by 9°C. Multiple sequence alignment explains the stabilizing effect of the mutated residues in the IL-24 variants by their presence in the related and more stable cytokines IL-20 and IL-19. Our homology-based approach can enhance existing methods for protein engineering and represents a viable alternative to study and produce difficult proteins for which only in silico structural information is available, estimated as >40% of all important drug targets.

An enhanced yeast display platform demonstrates the binding plasticity under various selection pressures

“…To further improve the protein’s stability, we combined computational and experimental procedures. Multiple stabilizing mutations were predicted by the consensual design of all available structures using the Pross web-server (Goldenzweig et al, 2016; Zahradnik et al, 2019). Since Pross was not designed to optimize secreted/surface-exposed proteins, we used the Pross suggested mutations as a starting point for random incorporation and selection, rather than testing suggested protein variants.…”

“…Table 1 also shows the plasmids constructed to complement our yeast display vectors with vectors for the production of fluorescent proteins: ALFA-tagged fluorescent proteins and fluorescent proteins fused with DnbALFA. The expression vectors are based on pET28bdSUMO vector (Zahradnik et al, 2019) and enable for bdSUMO protease single step, on-column cleavage based, purifications (Frey and Görlich, 2014). An example of proteins purified by this single step purification process is shown in Figure S3.…”

“…Protein expressions based on pET26b and pET28bdSUMO (Zahradnik et al, 2019) were done by using E. coli BL21(DE3) cells and 200 ml 2YT media (1L Erlenmeyer flasks). Cell cultures were grown (30°C, 220 rpm) to OD600 = 0.6, then the temperature was lowered to 20°C, protein expression was induced by 0.5 mM IPTG, and growth continued for 12 – 16h.…”

“…No template genes were used for the construction of these libraries. In contrast, the Kan-Nfr, sso7d, and s3LYV libraries were amplified from template DNA (pET28bdSUMO plasmids (Zahradnik et al, 2019)). In order to reduce the possibility of template amplification, the DNA was gel purified between each PCR extension step.…”

Stable and functionally diverse versatile peroxidases by computational design directly from sequence

“…In a number of cases, proteins that could not be functionally expressed in microbial systems could be expressed robustly after one-shot design calculations 17–19 . PROSS is sensitive to atomic details, however, and except in a handful of cases 20,21 , has not been applied to structural models.…”