Binding of small molecules to an adaptive protein-protein interface

Abstract

Understanding binding properties at protein-protein interfaces has been limited to structural and mutational analyses of natural binding partners or small peptides identified by phage display. Here, we present a high-resolution analysis of a nonpeptidyl small molecule, previously discovered by medicinal chemistry [Tilley, J. W., et al. (1997) J. Am. Chem. Soc. 119, 7589-7590], which binds to the cytokine IL-2. The small molecule binds to the same site that binds the IL-2 alpha receptor and buries into a groove not seen in the free structure of IL-2. Comparison of the bound and several free structures shows this site to be composed of two subsites: one is rigid, and the other is highly adaptive. Thermodynamic data suggest the energy barriers between these conformations are low. The subsites were dissected by using a site-directed screening method called tethering, in which small fragments were captured by disulfide interchange with cysteines introduced into IL-2 around these subsites. X-ray structures with the tethered fragments show that the subsite-binding interactions are similar to those observed with the original small molecule. Moreover, the adaptive subsite tethered many more compounds than did the rigid one. Thus, the adaptive nature of a protein-protein interface provides sites for small molecules to bind and underscores the challenge of applying structure-based design strategies that cannot accurately predict a dynamic protein surface.

Scheme 1

Structure of Compound 1.

Figure 1

(a) Compound 1/IL-2 complex determined by x-ray crystallography (Table 1). Compound 1 is shown in green sticks, IL-2 in white ribbon. The B–C loop is not defined by the electron density and is shown schematically in white spheres. (b) Interaction of Compound 1 (green sticks) with IL-2 (white sticks) taken from x-ray coordinates. Key contact side chains are labeled; H-bonds and distances are shown by yellow dotted lines. All molecular graphic images were produced with PYMOL (W. L. DeLano, San Carlos, CA).

Figure 2

Adaptivity of the IL-2-binding surface. (a) Structure of the complex of IL-2 and Compound 1, shown as a surface representation of IL-2 (white and orange) and stick representation of Compound 1 (green). The residues that comprise the IL-2Rα-binding hot spot are shown in orange. Residues contacting the molecule are labeled. (b–d) Surface representation of three different unliganded structures (21) (b) and Native I and II (c and d; Table 1). Compound 1 is overlaid to emphasize the extent of rearrangement that occurs in both of the unliganded structures and in the liganded form. Movie 1 (RIGIMOL, W. L. DeLano) highlights the dynamic nature of this site across this series of structures.

Figure 3

van't Hoff analysis of Compound 1 binding to IL-2. K_d values were determined by SPR at four temperatures (see Methods). (ΔH = −8.9 ± 0.35 kcal/mol, ΔS =−6.8 ± 0.56 cal/mol K.) Data are an average of three measurements; the standard deviations for each temperature are shown on the graph.

Scheme 2

Tethering method for fragment assembly.

Figure 4

Results from tethering experiments at cysteine variants surrounding the IL-2 hot spot. (a) The cysteine mutations located near the flexible region (shown in red; N30C, Y31C, N33C, L72C, and N77) selected >20 tethered fragments, whereas those made around the more ordered region of the protein (shown in blue; R38C, F42C, K43C, and Y45C) selected fewer than seven fragments. (b) The number of tether hits (z axis) versus the cysteine mutant position (y axis) and each of seven different tether compound libraries, each containing ≈1,000 compounds (x axis). The chemical structures represented in this graph are published as Table 3 in supporting information on the PNAS web site.

Figure 5

Tethering hits (in yellow) in the hydrophobic and hydrophilic subsites of IL-2 in comparison with Compound 1 (overlaid in green). The chemical structure of the fragment is shown above the structure. (a) X-ray structure of IL-2 with the indole glyoxylate fragment tethered at Y31C (Y31C/tether, Table 1). (b) X-ray structure of one of the guanidine tethering hits at K43C (K43C/tether). Movie 2, which is published as supporting information on the PNAS web site, highlights the different protein conformations.