Multi-State Design of Flexible Proteins Predicts Sequences Optimal for Conformational Change

Abstract

Computational protein design of an ensemble of conformations for one protein - i.e., multi-state design - determines the side chain identity by optimizing the energetic contributions of that side chain in each of the backbone conformations. Sampling the resulting large sequence-structure search space limits the number of conformations and the size of proteins in multi-state design algorithms. Here, we demonstrated that the REstrained CONvergence (RECON) algorithm can simultaneously evaluate the sequence of large proteins that undergo substantial conformational changes, such as viral surface glycoproteins. Simultaneous optimization of side chain conformations across all conformations resulted in an increase of 30% to 40% in sequence conservation when compared to single-state designs. More importantly, the sampled sequence space of RECON designs resembled the evolutionary sequence space of functional proteins. This finding was especially true for sequence positions that require substantial changes in their local environment across an ensemble of conformations. To quantify this rewiring of contacts at a certain position in sequence and structure, we introduced a new metric designated contact proximity deviation that enumerates contact map changes. This measure allows mapping of global conformational changes into local side chain proximity adjustments, a property not captured by traditional global similarity metrics such as RMSD or local similarity metrics such as changes in {varphi} and {psi} angles.\n\nAuthor SummaryMulti-state design can be used to engineer proteins that need to exist in multiple conformations or that bind to multiple partner molecules. In essence, multi-state design selects a compromise of protein sequences that allow for an ensemble of protein conformations, or states, associated with a particular biological function. In this paper, we used the REstrained CONvergence (RECON) algorithm with Rosetta to show that multi-state design of flexible proteins predicts sequences optimal for conformational change, mimicking mutation preferences sampled in evolution. Modeling optimal local side chain physicochemical environments within an ensemble selected significantly more native-like sequences than selections performed when all conformations states are designed independently. This outcome was particularly true for amino acids whose local side chain environment change between conformations. To quantify such contact map changes, we introduced a novel metric to show that sequence conservation is dependent on protein flexibility, i.e., changes in local side chain environments between stated limit the space of tolerated mutations. Additionally, such positions in sequence and structure are more likely to be energetically frustrated, at least in some states. Importantly, we showed that multi-state design over an ensemble of conformations (space) can explore evolutionary tolerated sequence space (time), thus enabling RECON to not only design proteins that require multiple states for function but also predict mutations that might be tolerated in native proteins but have not yet been explored by evolution. The latter aspect can be important to anticipate escape mutations, for example in pathogens or oncoproteins.