A comparison of successful and failed protein interface designs highlights the challenges of designing buried hydrogen bonds

Abstract

The accurate design of new protein-protein interactions is a longstanding goal of computational protein design. However, most computationally designed interfaces fail to form experimentally. This investigation compares five previously described successful de novo interface designs with 158 failures. Both sets of proteins were designed with the molecular modeling program Rosetta. Designs were considered a success if a high-resolution crystal structure of the complex closely matched the design model and the equilibrium dissociation constant for binding was less than 10 μM. The successes and failures represent a wide variety of interface types and design goals including heterodimers, homodimers, peptide-protein interactions, one-sided designs (i.e., where only one of the proteins was mutated) and two-sided designs. The most striking feature of the successful designs is that they have fewer polar atoms at their interfaces than many of the failed designs. Designs that attempted to create extensive sets of interface-spanning hydrogen bonds resulted in no detectable binding. In contrast, polar atoms make up more than 40% of the interface area of many natural dimers, and native interfaces often contain extensive hydrogen bonding networks. These results suggest that Rosetta may not be accurately balancing hydrogen bonding and electrostatic energies against desolvation penalties and that design processes may not include sufficient sampling to identify side chains in preordered conformations that can fully satisfy the hydrogen bonding potential of the interface.

Figure 1

Examples of successful (left) and unsuccessful (right) protein interface design models. Separate chains of successful designs are shown in purple and gray; the different chains of failed models are colored green and brown; dashed black lines represent interface spanning side-chain involved hydrogen bonds. The structures shown represent examples design models of β-strand mediated interface (A, B),two models targeting flu HA (C, D), and design of helix secondary structure to bind a target protein. The successful models shown are βdimer1 (A), HB36 (C), and GL_helix-4 (D). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2

Interface energy density as computed by Rosetta for (△G_bind/△SASA) designed and natural interfaces. The change in SASA upon binding versus △G_bind/△SASA is shown for native heterodimers (A), native homodimers (B), and all designed interfaces(C). Large points with gray interiors represent the successful design models. Least-squares lines were fit to each set of interfaces. The correlation coefficient for the design models is r = 0.33.

Figure 3

Packing quality measure of the design models and Rosetta minimized natural interfaces. Two independent measures of packing quality are shown; (A) the shape complementarity score for the interface and (B) the RosettaHoles score for residues at the interface. For each metric a value of 1.0 represents perfect packing, while lower values represent packing defects. Lines represent the successful design models.

Figure 4

Polar content of designed and natural interfaces. The polar fraction of interface area is shown for designs versus heterodimers (A) and homodimers (B). Successful designs highlighted by lines. An asterisk above the line denotes the value for the crystal structure while no asterisk is present above the successful design models.

Figure 5

Buried polar atoms and buried hydrogen bonds at interfaces. Values for Rosetta minimized crystal structures are shown by lines. A: The number of buried polar atoms without a hydrogen-bonding partner per 1,000 Ų of interface area. B: The total energy of a buried, side-chain involved hydrogen bond at the interface as a fraction of total binding energy (△G_bind). HB36 and HB80 acquired an additional buried hydrogen bond due to mutations introduced by affinity maturation.