De Novo Design of a β-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer’s Disease

Abstract

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer’s disease (AD), where the spatio–temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a β-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen–Shannon Divergence and the embedding depth showed that the multi-epitope scaffolding approach, employed in designing the β-helix scaffold, was predicted to better discriminate toxic tau oligomers than other “monovalent” strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the β-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized β-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the β-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Cyclization Scaffolding for Improved Vaccine Immunogen Stability: Application to Tau Protein in Alzheimer’s Disease

“…All cyclized constructs have greater dynamic metastability than the uncyclized linear construct, which has an rmsd of 2.67 Å as shown by the top dashed line. Additionally, most of the cyclized designs have improved dynamic metastability over the corresponding native sequence in the context of the complete fibril (5O3L), as shown by the lower dashed line at 1.74 Å . The cyclized fibrils with three-residue linkers are more stable than the cyclized fibrils with four residue linkers.…”

“…Our objective is to use a rational approach to design a vaccine immunogen that structurally resembles the toxic tau oligomer for a specific targeted epitope. Previously, we proposed oligomer-selective β-helix scaffolds that increase the fibrillar conformational stability of a disorder-prone region in the tau fibril structure (5O3L), 339 VKSEKLDFKDRVQSKI 354 (Figure A) . Here, we present an alternative method that stabilizes the local structure of this segment, by head-to-tail peptide bond cyclization of each chain in a multichain complex.…”

“…This is confirmed by tracking backbone hydrogen bonds in Rosetta, indicating an average of 3.4 hydrogen bonds per linker. The representative structures for clusters DDL and 42 The cyclized fibrils with three-residue linkers are more stable than the cyclized fibrils with four residue linkers. The p-value indicates a statistically significant difference between the stability of cyclized peptide fibrils with three-residue linkers compared to those with four-residue linkers.…”

“…We therefore proposed an alternative scaffolding method, wherein multiple copies of the epitope hypothesized to be present in the protofibril were patched into a single chain with a beta-helix structure to mimic an oligomer-like conformation for that epitope. This approach shows promise for the tau protein: Antibody binding tests using surface plasmon resonance support the β-helix immunogen as presenting an oligomer-like conformation for the epitope . We emphasize that this strategy takes a disorder-prone region of the protofibril and then seeks to scaffold it by inducing to adopt a more fibril-like structure.…”

“…We have previously identified 343 KLDFK 348 as a candidate for an oligomer-selective epitope using the Collective Coordinates computational platform. , This region is the most weakly stable part of the fibril, and thus, it has to be properly scaffolded in order to present the epitope in an oligomer-like protofibril conformation.…”