Human IL-2Rɑ subunit binding modulation of IL-2 through a decline in electrostatic interactions: A computational and experimental approach

Abstract

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rβγc subunits interactions.

Fig 1

A. IL-2 protein structure (hot spot residues in sticks), B. Back-bone root mean square fluctuations (RMSD) of the native (green), mutant 1 (blue) and mutant 2 (magenta) protein, C. Per-residue root mean square fluctuations (RMSF) of the native (green), mutant 1 (blue) and mutant 2 (magenta) protein residues, D. Per-residue root mean square fluctuations (RMSF) of the target residues 30–50 close view, E. Per-residue root mean square fluctuations (RMSF) of the target residues 55–75 close view, F. Residues F42, F43 and P65, and g. Residues L72 and R38.

Fig 2. Secondary structure analysis.

A. The native protein, B. Mutant 1 and, C. Mutant 2, during 100 ns of MDs (where the structure is shown in blue, α-helix in dark blue, 3-helix in gray, coil in orange, turn in green, β-sheet in magenta, b-bridge in yellow, and bend in cyan), D. α-helix structure of native structure (green), mutant 1(blue) and mutant 2 (magenta), E. Coil structure of native structure (green), mutant 1(blue) and mutant 2 (magenta), F. Turn structure of native structure (green), mutant 1(blue) and mutant 2 (magenta), G. superimposition of the native IL-2 and IL-2Rα interacting residues over mutant 1, and H. superimposition of mutant 2 and IL-2Rα interacting residues over mutant 2 IL-2.

Fig 3. Back-bone root mean square fluctuations (RMSD).

A. IL-2Rβ (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins), B. IL-2Rγc (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins), C. IL-2Rα (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins), and D. Native IL-2 (green), mutant 1 (blue) and mutant 2 (magenta) proteins) during 100ns of MDs. Back-bone root mean square fluctuations (RMSF) of, E. IL-2Rβ residues (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins, F. γc residues (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins), G. IL-2Rα residues (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins), and H. wtIL-2 residues (green), mutant 1 (blue) and mutant 2 (magenta) during 100ns of MDs.

Fig 4

Interactions between A. wtIL-2 and IL-2Rαβγc, B. M1 and IL-2Rαβγc, and C. M2. IL-2Rαβγc after 100 ns of MDs, as well as the close view of D. wtIL-2 and IL-2Rα interactions, E. M1 and IL-2Rα and F. M2 and IL-2Rα. The hydrogen bonds are shown in yellow dashes. The red and blue spheres represent charging interactions (salt bridges), and the yellow spheres represent electrostatic (like π-π, alkyl-π and π-cation) interactions.

Fig 5. Snapshots of umbrella sampling simulations of the target complex.

A. Native IL-2, B. Mutant 1and C. Mutant 2. D. Potentials of mean forces (PMF) for IL-2 and IL-2Rα (the native (green), mutant 1 (blue) and mutant 2 (magenta) proteins) as a function of the reaction coordinate (ξ).

Fig 6. SDS-PAGE and Western blotting analysis of the recombinant proteins.

Lane 1, protein marker (10–250 kDa). Whole lysate of BL21(DE3)-pET-22b without gene (lane 2), expressed wtIL-2 (lane3), mutant IL-2 (lane4).

Fig 7. Flow cytometry analysis showing the binding ability of IL-2 mutein to the conA activated PBMC.

A. Direct binding, red graph negative control, green graph wtIL-2 (left panel) and mutant IL-2 (right panel) bound to the cells and detected with rabbit anti-His-tag Ab and mouse anti-rabbit FITC-conjugated. B. Flow cytometry analysis showing the competitive assay. Negative control (red graph), cells labeled with anti-CD25 MAb-PE (green), and cells previously incubated with wtIL-2 (blue graph; left panel) or mutant IL-2 (blue graph; right panel) and then labeled with the anti-CD25 MAb-FITC.