Rosetta FunFolDes – A general framework for the computational design of functional proteins

Abstract

The robust computational design of functional proteins has the potential to deeply impact translational research and broaden our understanding of the determinants of protein function and stability. The low success rates of computational design protocols and the extensive in vitro optimization often required, highlight the challenge of designing proteins that perform essential biochemical functions, such as binding or catalysis. One of the most simplistic approaches for the design of function is to adopt functional motifs in naturally occurring proteins and transplant them to computationally designed proteins. The structural complexity of the functional motif largely determines how readily one can find host protein structures that are “designable”, meaning that are likely to present the functional motif in the desired conformation. One promising route to enhance the “designability” of protein structures is to allow backbone flexibility. Here, we present a computational approach that couples conformational folding with sequence design to embed functional motifs into heterologous proteins—Rosetta Functional Folding and Design (FunFolDes). We performed extensive computational benchmarks, where we observed that the enforcement of functional requirements resulted in designs distant from the global energetic minimum of the protein. An observation consistent with several experimental studies that have revealed function-stability tradeoffs. To test the design capabilities of FunFolDes we transplanted two viral epitopes into distant structural templates including one de novo “functionless” fold, which represent two typical challenges where the designability problem arises. The designed proteins were experimentally characterized showing high binding affinities to monoclonal antibodies, making them valuable candidates for vaccine design endeavors. Overall, we present an accessible strategy to repurpose old protein folds for new functions. This may lead to important improvements on the computational design of proteins, with structurally complex functional sites, that can perform elaborate biochemical functions related to binding and catalysis.

Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen

“…Whereas the RSV antigenic site II is a structurally simple helix-turn-helix motif, many other identified neutralization epitopes comprise multiple, discontinuous segments. However, continuous advances in rational protein design techniques [65] will allow the design of more complex protein scaffolds to stabilize increasingly complex epitopes.…”

De novo protein design enables the precise induction of RSV-neutralizing antibodies

“…In a first effort, we used a template-based de novo design approach relying on Rosetta FunFolDes (34) to fold and design scaffolds for sites IV and 0. Given the structural complexity of these sites, few structures in the Protein Data Bank (PDB) matched the backbone con-figuration of the epitopes, even using loose structural criteria ( fig.…”

“…The final stage is flexible backbone sampling and sequence design. To refine the structural features of the sketches at the all-atom level and to design sequences that stabilize these structures, we used Rosetta FunFolDes as described previously (9,34).…”

rstoolbox - a Python library for large-scale analysis of computational protein design data and structural bioinformatics

“…Recently, we developed the Rosetta FunFolDes protocol, which was devised to couple conformational folding and sequence design [28]. FunFolDes was developed to insert functional sites into protein scaffolds and allow for full-backbone flexibility to enhance sequence sampling.…”