Rosetta Design With Co-Evolutionary Information Retains Protein Function

Author summaryIn homologous proteins, functionally or structurally important residues are strongly conserved. Thus, the consideration of conservation signals during protein design protocols can help to create sequences that are more native-like. However, the number of conserved residues is small in many proteins and not all important residues can be captured by conservation analysis. Residues are forming networks whose composition is dictated by protein structure function and thus is visible through the co-evolutionary analysis. Nowadays, advanced methods allow us to deduce these networks from multiple sequence alignments. Thus, we have implemented the novel Rosetta method termed 'ResCue' that informs the design protocol with co-evolutionary signals. Recapitulation designs based on ten difficult benchmarks made clear that this protocol creates sequences that are more native-like than three other, state-of-the-art design protocols.Computational protein design has the ambitious goal of crafting novel proteins that address challenges in biology and medicine. To overcome these challenges, the computational protein modeling suite Rosetta has been tailored to address various protein design tasks. Recently, statistical methods have been developed that identify correlated mutations between residues in a multiple sequence alignment of homologous proteins. These subtle inter-dependencies in the occupancy of residue positions throughout evolution are crucial for protein function, but we found that three current Rosetta design approaches fail to recover these co-evolutionary couplings. Thus, we developed the Rosetta method ResCue (residue-coupling enhanced) that leverages co-evolutionary information to favor sequences which recapitulate correlated mutations, as observed in nature. To assess the protocols via recapitulation designs, we compiled a benchmark of ten proteins each represented by two, structurally diverse states. We could demonstrate that ResCue designed sequences with an average sequence recovery rate of 70%, whereas three other protocols reached not more than 50%, on average. Our approach had higher recovery rates also for functionally important residues, which were studied in detail. This improvement has only a minor negative effect on the fitness of the designed sequences as assessed by Rosetta energy. In conclusion, our findings support the idea that informing protocols with co-evolutionary signals helps to design stable and native-like proteins that are compatible with the different conformational states required for a complex function.