Specificity versus stability in computational protein design

Abstract

Protein-protein interactions can be designed computationally by using positive strategies that maximize the stability of the desired structure and͞or by negative strategies that seek to destabilize competing states. Here, we compare the efficacy of these methods in reengineering a protein homodimer into a heterodimer. The stability-design protein (positive design only) was experimentally more stable than the specificity-design heterodimer (positive and negative design). By contrast, only the specificity-design protein assembled as a homogenous heterodimer in solution, whereas the stability-design protein formed a mixture of homodimer and heterodimer species. The experimental stabilities of the engineered proteins correlated roughly with their calculated stabilities, and the crystal structure of the specificity-design heterodimer showed most of the predicted side-chain packing interactions and a mainchain conformation indistinguishable from the wild-type structure. These results indicate that the design simulations capture important features of both stability and structure and demonstrate that negative design can be critical for attaining specificity when competing states are close in structure space.adaptor protein ͉ protein engineering ͉ SspB H ighly specific recognition lies at the core of most cellular processes. The interaction of two partner proteins in a crowded intracellular environment depends on the equilibrium stability of the complex, which is determined by affinity and concentration, but is also controlled by the competing binding of either partner to other cellular macromolecules. Efficient protein recognition requires both stability and specificity. In natural systems, both parameters are subject to evolutionary optimization. Likewise in engineered systems, stability can be targeted for maximization, and known competing states or interactions can be minimized through the use of negative design. The biophysical tradeoff between stability and specificity in molecular recognition is a fascinating problem that has important implications in the development of practical tools for synthesizing and dissecting biological systems.Focusing on stability without explicit consideration of specificity has resulted in impressive protein-engineering feats, including full-sequence design of a zinc-finger protein that folds in the absence of metal (1), introduction of catalytic activity into previously inert protein scaffolds (2, 3), and the creation of a novel protein fold (4). These successes relied on positive design only. Positive design maximizes favorable interactions in the target conformation. Negative design, by contrast, maximizes unfavorable interactions in competing states and requires modeling of each unwanted conformation (5, 6).Protein-protein interactions have been successfully reengineered to alter binding specificity both by using and ignoring negative design (7-15). How is success possible in the absence of negative design? One possibility is that most changes that optimize the stability of a targe...

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