Volume 10, Issue 7, Pages 981-987 (July 2002) Noncompetitive Antibody Neutralization of IL-10 Revealed by Protein Engineering and X-Ray Crystallography  Kristopher Josephson, Brandi C Jones, Leigh J Walter, Ruth DiGiacomo, Stephen R Indelicato, Mark R Walter  Structure  Volume 10, Issue 7, Pages 981-987 (July 2002) DOI: 10.1016/S0969-2126(02)00791-8

Figure 1 The IL-10M1/9D7Fab Complex and Interface (A) Ribbon diagram of the IL-10M1/9D7Fab complex with IL-10M1 in orange and the 9D7Fab heavy and light chains in red and blue, respectively. The 9D7Fab epitope, residues 71–83 and 125–137, is in green. (B) Stereo diagram of the IL-10M1/9D7Fab interface colored as described in panel A. Nitrogen atoms, blue; oxygen atoms, red; interfacial waters, magenta spheres; hydrogen bonds, dashed black lines. Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)

Figure 2 Location of 9D7Fab and IL-10R1 Binding Sites and IL-10 Structural Changes (A) Ribbon diagram of the ternary complex model between IL-10 (green), sIL-10R1 (yellow), and 9D7Fab (blue and red). Two 9D7Fab fragments were positioned by superimposing IL-10M1 from the IL-10M1/9D7Fab complex on each of the domains in the 1:2 IL-10/sIL-10R1 complex (Protein Data Bank code 1J7V). (B) The upper histogram plot displays the buried surfaces per residue in the interfaces of the IL-10M1/9D7Fab (red) and IL-10/sIL-10R1 (Protein Data Bank code 1J7V; yellow) complexes. The secondary structure is displayed below with α helices represented by gray boxes and loops by thin black lines. The lower histogram plot displays the rmsd between Cα atoms of 9D7Fab-bound IL-10M1 and receptor-bound IL-10 (Protein Data Bank code 1J7V; green) or 9D7Fab-bound IL-10M1 and free IL-10 (Protein Data Bank code 2ILK; magenta). Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)

Figure 3 Structural Changes in the IL-10M1 AB Loop (A) Stereo view of the electron density for the residues 36–45 in IL-10M1 corresponding to helix A and the AB loop. The Fo − Fc map is contoured at 1.7 σ and was calculated from six simulated-annealing models from which residues 35–47 had been removed. (B) Stereo view ribbon diagram comparing IL-10M1 (orange) and receptor-bound IL-10 (Protein Data Bank code 1J7V; green). The 9D7Fab heavy and light chains are red and blue, respectively, with selected side chains in gray. Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)

Figure 4 Effect of 9D7Fab-Induced Changes on Receptor Binding (A) Comparison of helix A and AB loop residues for 9D7-bound IL-10M1 (orange) and receptor-bound IL-10 (Protein Data Bank code 1J7V; green). Helix F, which sits behind the AB loop, has been removed for clarity. Hydrogen bonds between IL-10 and sIL-10R1 (yellow side chains) in the IL-10/sIL-10R1 receptor complex are indicated. The side chain of IL-10R1 residue R76 has two conformations. (B) ITC analysis of the interaction between IL-10M1 and sIL-10R1 in the presence of 9D7Fab (orange) compared to the identical experiment lacking 9D7Fab (green). The top panel contains the raw data for both experiments, and the bottom panel contains a plot of the heat changes against the ratio of sIL-10R1 to IL-10M1. Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)

Figure 5 Isolation of the Ternary IL-10M1/sIL-10R1/9D7Fab Complex (A) Analysis of the complexes formed when IL-10M1, sIL-10R1, and 9D7Fab are mixed at 250 μM by size exclusion chromatography. (B) Nonreducing SDS-PAGE analysis of the peaks in (A). Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)

Figure 6 Stereo Model Showing How 9D7Fab Prevents the Formation of the 2 IL-10/4 sIL-10R1 Complex The 2 IL-10 dimer/4 sIL-10R1 complex has been proposed as a model for the active IL-10/IL-10R1/IL-10R2 complex, constructed by the replacement of the sIL-10R1 chains bound to one IL-10 dimer (red) with IL-10R2 (blue) [6]. In this model, activation occurs between IL-10R1 and IL-10R2 bound to adjacent IL-10 dimers, rather than across them. Steric clashes between the magenta and cyan 9DFab molecules bound to the green and the red IL-10 dimers, respectively, would disrupt the formation of the “active” 2 IL-10 dimer/2 IL-10R1/2 IL-10R2 complex. Structure 2002 10, 981-987DOI: (10.1016/S0969-2126(02)00791-8)