Functionalization of a symmetric protein scaffold: Redundant folding nuclei and alternative oligomeric folding pathways

Abstract

Successful de novo protein design ideally targets specific folding kinetics, stability thermodynamics, and biochemical functionality, and the simultaneous achievement of all these criteria in a single step design is challenging. Protein design is potentially simplified by separating the problem into two steps: (a) an initial design of a protein “scaffold” having appropriate folding kinetics and stability thermodynamics, followed by (b) appropriate functional mutation—possibly involving insertion of a peptide functional “cassette.” This stepwise approach can also separate the orthogonal effects of the “stability/function” and “foldability/function” tradeoffs commonly observed in protein design. If the scaffold is a protein architecture having an exact rotational symmetry, then there is the potential for redundant folding nuclei and multiple equivalent sites of functionalization; thereby enabling broader functional adaptation. We describe such a “scaffold” and functional “cassette” design strategy applied to a β‐trefoil threefold symmetric architecture and a heparin ligand functionality. The results support the availability of redundant folding nuclei within this symmetric architecture, and also identify a minimal peptide cassette conferring heparin affinity. The results also identify an energy barrier of destabilization that switches the protein folding pathway from monomeric to trimeric, thereby identifying another potential advantage of symmetric protein architecture in de novo design.

Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity

“…Thus, optimization of core interactions for desired kinetic stability can provide a valuable tool for practical engineering of functional proteins. In addition, developing a scaffold with high kinetic stability may provide a particularly useful starting point for subsequent engineering of function, which often impairs stability ( Liu et al, 2001 ; Gosavi, 2013 ; Tenorio et al, 2022 ). Core residue engineering may be even more accessible for proteins containing repetitive structures and symmetry such as the structurally symmetric β-trefoils and TIM barrels, or repeat proteins ( Meiering et al, 1991 ; Sancho et al, 1991 ; Broom et al, 2015b ; Broom et al, 2016 ; Vrancken et al, 2020 ).…”

“…Frontiers in Molecular Biosciences frontiersin.org engineering of function, which often impairs stability (Liu et al, 2001;Gosavi, 2013;Tenorio et al, 2022). Core residue engineering may be even more accessible for proteins containing repetitive structures and symmetry such as the structurally symmetric β-trefoils and TIM barrels, or repeat proteins (Meiering et al, 1991;Sancho et al, 1991;Broom et al, 2015b;Broom et al, 2016;Vrancken et al, 2020).…”

Variable and Conserved Regions of Secondary Structure in the β-Trefoil Fold: Structure Versus Function

“…Overall, the β-trefoil architecture has many attractive features for de novo protein design, applied especially to ligand functionality. The adoption of heparin-binding functionality into a benign β-trefoil scaffold using the principles described herein has recently been demonstrated ( Tenorio et al, Forthcoming 2022 ).…”