Extended Data Fig. 3: Single disulfide-stapled variants of Neo-2/15 with higher thermal stability. | Nature

Extended Data Fig. 3: Single disulfide-stapled variants of Neo-2/15 with higher thermal stability.

From: De novo design of potent and selective mimics of IL-2 and IL-15

Extended Data Fig. 3

a, b, Structural models of disulfide-stabilized variants of Neo-2/15 (grey) are shown superposed on the ternary crystal structure of Neo-2/15 (red) with mutated residues highlighted in magenta and the disulfide bond shown in gold. Two strategies were used to generate the disulfide stapled variants. a, Top, internal placement of the disulfide linking residues 38 and 75. Bottom, experimental CD spectra of the design at 25 °C, 95 °C and then cooled back to 25 °C, showing complete recovery of ellipticity spectrum (full reversibility) upon cooling. b, Top, for the terminal disulfide variant, three residues were added to each terminus in order to allow the disulfide to be formed without distorting the Neo-2/15 structure. Bottom, experimental CD spectra of the design at 25 °C, 95 °C and then cooled back to 25 °C, showing complete recovery of ellipticity spectrum (full reversibility) upon cooling. c, Thermal melting of each disulfide variant in a and b between 25 °C and 95 °C (heating rate ≈ 2 °C min−1) was monitored using circular dichroism at 222 nm. Each of the disulfide-stapled variants shows improved stability relative to native Neo-2/15. d, Binding strength of each disulfide variant was measured by biolayer interferometry, showing that the introduction of disulfide bonds does not disrupt binding. Furthermore, both disulfide variants exhibit improved binding of IL-2Rβγc (Kd ≈ 1.3 ± 0.49 nM and 1.8 ± 0.26 nM for the internal and external disulfide staples, respectively), compared to Neo-2/15 (Kd ≈ 6.9 ± 0.61 nM) under the same experimental conditions. These results are consistent with the expected effect of disulfide-induced stabilization on a de novo protein binding site71. Thermal denaturation experiments were performed 3 times with similar results; binding experiments were performed once.