A structure-based benchmark for protein-protein binding affinity

Abstract

We have assembled a nonredundant set of 144 protein-protein complexes that have high-resolution structures available for both the complexes and their unbound components, and for which dissociation constants have been measured by biophysical methods. The set is diverse in terms of the biological functions it represents, with complexes that involve G-proteins and receptor extracellular domains, as well as antigen/antibody, enzyme/inhibitor, and enzyme/substrate complexes. It is also diverse in terms of the partners' affinity for each other, with K(d) ranging between 10(-5) and 10(-14) M. Nine pairs of entries represent closely related complexes that have a similar structure, but a very different affinity, each pair comprising a cognate and a noncognate assembly. The unbound structures of the component proteins being available, conformation changes can be assessed. They are significant in most of the complexes, and large movements or disorder-to-order transitions are frequently observed. The set may be used to benchmark biophysical models aiming to relate affinity to structure in protein-protein interactions, taking into account the reactants and the conformation changes that accompany the association reaction, instead of just the final product.

Figure 1

Cognate and noncognate colicin/immunity protein complexes. The DNase domain of colicin E9 (cyan) has a very high affinity for the Im9 immunity protein produced by the same E. coli strain (green), and a 10⁶-fold lower affinity for Im2, produced by a different strain. The crystal structures of the cognate E9/Im9 (1EMV) and the noncognate E9/Im2 (2WPT) complexes indicate that the mode of assembly is essentially the same. The insert shows that, nevertheless, segment 22–30 of Im9 (red) interacting with Tyr83 of E9 undergoes a significant movement in Im2 (blue), where it has a different sequence.

Figure 2

Allostery and ligand affinity. Protein A binds ligands B and C at two distinct sites that have different affinities in the binary and the ternary complexes. The ratio K₁/K₃ = K₄/K₂ is a measure of the cooperative interaction between the ligands. The table reports dissociation constants (in molar units); they are from experiment, except for K₃ in 2TPI, which is an estimate based on trypsin, and the three values in bold face, calculated from the linkage equation; the values retained for the benchmark are underlined. References: 2TPI, 1H9D,, and 1K5D.

Figure 3

Conformation changes and binding free energy. Assuming ΔG_calc = α ΔASA + β, a linear regression of the observed ΔG's vs. the interface area ΔASA was performed on 70 complexes with I_rmsd < 1 Å (filled circles) excluding two (1BRS and 2PTC). The regression yields R = 0.54 and a RMS discrepancy between ΔG_calc and ΔG_obs of 2.4 kcal mol⁻¹. When I_rmsd > 1 Å, the correlation with ΔASA vanishes, and 70% of the points are below the diagonal, meaning that observed ΔG values are less than calculated ones (with P < 10⁻⁴). The average value of ΔG_calc − ΔG_obs is 1.2 kcal mol⁻¹ for 39 complexes with I_rmsd in the range 1–1.5 Å (crosses), and 2.7 kcal mol⁻¹ for 35 complexes with I_rmsd > 1.5 Å (empty circles).