Fig 1.
Template-based rescaffolding strategy.
3D functional descriptors representing the side chain functionalities of selected IL-10R1 binding residues (yellow sticks) are used to define the 3D pattern syntax query R-<2,6>-R-<4,5>-Y- to search for seeding templates in PDB. One of the best hits, used as seeding template (2ACA149-157, in green), is shown superimposed to the selected IL-10R1 relevant residues for molecular recognition. Molecular images created with PyMOL [29].
Table 1.
Residues in PDB matching the 3D pattern syntax query R-<2,6>-R-<4,5>-Y-.
They are underlined in their corresponding PDB structural motifs.
Fig 2.
Structure-based design rationale.
(A) Atomic representation of the model of the complex between IL-10 (gray, PDB ID 1J7V) and the helical scaffold used in the rationale for the design of IL-10R1 mimetics (green, N-terminal functionalization in yellow). For clarity, no side chains shown for IL-10Q38 and IL-10L48. (B) Snapshot of complex IL-10/M1 (at 2.9 ns from 10 ns MD simulation). (C) Snapshot of complex IL-10/M4 (at 10 ns). (D) Snapshot of complex IL-10/M6 (at 3.6 ns). Residues involved in recognition are shown in sticks and colored by atom type (IL-10 highlighted in violet in A). Red spheres represent interfacial structural waters observed during MD simulations. Intermolecular H-bonds are depicted by black dashed lines. Figure generated in PyMOL [29].
Table 2.
Mimetic-protein binding free energy obtained by MM-PBSA [43, 44] and experimental dissociation constants (Kd).[a],[b]
Fig 3.
Temperature denaturation curves obtained by CD for mIL-10C149Y in complex with IL-10R1 mimetics M1-M4.
The average thermal denaturation CD signal in the 215 to 230 nm range is plotted as a function of temperature for mIL-10C149Y alone (2 μM, red open circles) and for mIL-10C149Y in the presence of each mimetic M1-M4 (4.5 μM, black filled squares) in panels A-D, respectively. The obtained mIL-10C149Y CD data agree with those published earlier for human and murine IL-10 [50]. The highest amount of native protein structure binding to each of the mimetics M1-M4 was observed at 332 K (dashed lines) and is given in each corresponding panel as percentage of the total change in the CD-signal.
Fig 4.
Temperature-dependent excitation spectra of the tryptophan in mimetics M1-M4.
Upper plots A-D: Mimetic tryptophan emission (excited between 200 and 290 nm) measured at 360 nm for the temperature range between 298 K and 363 K for each mimetic M1-M4 in the presence of mIL-10C149Y. The excitation spectra at 332 K are highlighted in red. Lower plots A-D: Subtraction of the excitation spectra at 298 K from those at 332 K (red) in comparison with the spectra obtained in absence of mIL-10C149Y (blue).
Fig 5.
Isothermal titration calorimetry.
(A) mIL-10C149Y (43 μM) with M5. (B) mIL-10C149Y (25 μM) with M6. Upper and lower panels show the raw data and integrated heat changes with the corresponding fitted binding curves based on a single site model, respectively.
Fig 6.
3D pattern syntax: Sequence patterns and structural connectors.