Bottom-up de novo design of functional proteins with complex structural features

Abstract

De novo protein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to build de novo proteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. The de novo proteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.

Multiepitope Proteins for the Differential Detection of IgG Antibodies against RBD of the Spike Protein and Non-RBD Regions of SARS-CoV-2

“…The major challenge was developing a method to bioengineer a multiepitope coding sequence that would result in a protein [ 39 , 40 ]. To maintain the interactions of the epitopes with antibodies, it was clear that the approach needed to maintain their sequences on the surface of the resulting protein.…”

RosettaSurf - a surface-centric computational design approach

“…The incorporation of surface similarity clearly increases sequence recovery rates relative to other scoring schemes. This approach can be readily applied for the design of experimental combinatorial libraries to reduce library sizes, as the high sequence recovery rates suggest that surface-centric design is especially interesting to design novel proteins that mimic surface patches of other proteins, as it is the case for the design of novel immunogens for vaccine development (36)(37)(38). We demonstrated that surface-centric design could be used to generate targeted libraries to optimize binding specificity of a previously reported IL-2 design (61).…”

“…By focusing on protein interfaces, the correct sequence does not solely depend on minimizing the Rosetta scoring function, but rather needs to represent the surface properties of the interface site. While such design scenario has limited applicability for the de novo design of protein-protein interactions, it is important for applications in the domain of immunogen design for vaccine development where the surface mimicry of known surfaces (neutralizing epitopes) is critical for the biological activity of this type of design (34,(36)(37)(38). In this benchmark we considered nine protein-protein complexes, grouped into three categories (low complementarity, high complementarity, antibody/antigen), and evaluated the performance by assessing sequence recovery (Fig.…”

“…Previous studies have shown that identifying scaffolds that can present such complex structural motifs is challenging, and specifically for site 0 they are currently not available in the known structure space. De novo design methods have been successfully used to build scaffolds from scratch (37,38), but the design process remains challenging and expertise in de novo protein design is critical. The RosettaSurf approach allows to rescue protein scaffolds that only present partial structural matches to the epitope structure motif.…”

“…by sampling different amino acid side chains and adjusting atoms in the protein structure to optimize a given energy function. While these strategies have been successfully applied to design novel protein topologies (28)(29)(30), the design of functional proteins based solely on computational calculations remains a challenge (31)(32)(33)(34)(35)(36)(37)(38). The introduction of function into a designer protein often requires the transplantation of functional sites by means of protein grafting (39)(40)(41)(42)(43)(44).…”