De Novo Design of a Highly Stable Ovoid TIM Barrel: Unlocking Pocket Shape towards Functional Design

Abstract

The ability to finely control the structure of protein folds is an important prerequisite to functional protein design. The TIM barrel fold is an important target for these efforts as it is highly enriched for diverse functions in nature. Although a TIM barrel protein has been designed de novo, the ability to finely alter the curvature of the central beta barrel and the overall architecture of the fold remains elusive, limiting its utility for functional design. Here, we report the de novo design of a TIM barrel with ovoid (twofold) symmetry, drawing inspiration from natural beta and TIM barrels with ovoid curvature. We use an autoregressive backbone sampling strategy to implement our hypothesis for elongated barrel curvature, followed by an iterative enrichment sequence design protocol to obtain sequences which yield a high proportion of successfully folding designs. Designed sequences are highly stable and fold to the designed barrel curvature as determined by a 2.1 Å resolution crystal structure. The designs show robustness to drastic mutations, retaining high melting temperatures even when multiple charged residues are buried in the hydrophobic core or when the hydrophobic core is ablated to alanine. As a scaffold with a greater capacity for hosting diverse hydrogen bonding networks and installation of binding pockets or active sites, the ovoid TIM barrel represents a major step towards the de novo design of functional TIM barrels.

Computational design of soluble functional analogues of integral membrane proteins

“…The TBF has been a long-standing challenge in protein design 47–49 , and only very recently, several studies successfully designed this fold 10,50,51 . Previous TBF design strategies imposed symmetry and parametric restraints both at the structural and sequence level 50,51 . With our pipeline, we were able to design TBFs without any constraints, allowing for a larger structural and sequence diversity, and even asymmetry, which could potentially accommodate more complex enzymatic sites (Extended Data Fig.…”

“…The TIM-barrel fold comprises eight parallel-paired β-strands, each separated by an alpha helix, resulting in long-range interactions between the β-strands 47 . The TBF has been a long-standing challenge in protein design [47][48][49] , and only very recently, several studies successfully designed this fold 10,50,51 .…”

De novodesign of buttressed loops for sculpting protein functions

“…Our approach alone or integrated with additional recent progress in loop design 16 and recently developed deep learning approaches for protein design [17][18][19][20][21] (which do not currently address the challenge of designing structured long loops), should enable the design of structured loops for binding functions and beyond in a wide variety of scaffolds. For example, for enzyme design, multiple loops emanating from designed TIM barrels [22][23][24] could be built to buttress each other and, together with the residues emerging from the top of the beta strands and helices in the TIM structure, form an extensive catalytic site and associated substrate/transition state binding site.…”

Opportunities and Challenges for Machine Learning-Assisted Enzyme Engineering

“…While most current success involves generating protein scaffolds or activities that are already known, it will be exciting to see more efforts that focus on generating enzymes that do not resemble those in nature and/or exhibit non-natural activities. In protein engineering, certain protein folds are more evolvable for certain reasons, including elevated stability , that is imparted by residues outside the active site, , balanced with flexibility to change conformation and accommodate new substrates and reactions . Proteins that express well in a host organism for evolution are also preferred.…”