Reconfigurable Peptide Analogs of Apolipoprotein A-I Reveal Tunable Features of Nanodisc Assembly

Abstract

Nanodisc (ND)-forming membrane scaffold proteins or peptides developed from apolipoprotein A-I (apoA-I) have led to considerable promise in structural biology and therapeutic applications. However, the rationale and regularity characteristics in peptide sequence design remain inconclusive. Here, we proposed a consensus-based normalization approach through the reversed engineering of apoA-IΔ1–45 to design reconfigurable apoA-I peptide analogs (APAs) for tunable ND assembly. We present extensive morphological validations and computational simulation analyses on divergent APA-NDs that are generated by our method. Fifteen divergent APAs were generated accordingly to study the assembly machinery of NDs. We show that APA designs exhibit multifactorial influence in terms of varying APA tandem repeats, sequence composition, and lipid-to-APA ratio to form tunable diameters of NDs. There is a strong positive correlation between DMPC-to-APA ratios and ND diameters. Longer APA with more tandem repeats tends to yield higher particle size homogeneity. Our results also suggest proline is a dispensable residue for the APA-ND formation. Interestingly, proline-rich substitution not only provides an inward-bending effect in forming smaller NDs but also induces the cumulative chain flexibility that enables larger ND formation at higher lipid ratios. Additionally, proline-tryptophan residues in APAs play a dominant role in forming larger NDs. Molecular simulation shows that enriched basic and acidic residues in APAs evoke abundant hydrogen-bond and salt bridge networks to reinforce the structural stability of APA-NDs. Together, our findings provide a rational basis for understanding APA design. The proposed model could be extended to other apolipoproteins for desired ND engineering.

Accelerating therapeutic protein design with computational approaches toward the clinical stage

“…In addition to exploring the mechanism of stability, molecular dynamics can also be used to design protein therapeutics with improved stability [208] . In our latest work, we developed a consensus-based normalization approach for designing reconfigurable apoA-I peptide analogs (APAs) to create tunable ND assemblies, with potential implications for structural biology and therapeutics [209] . We generated 15 divergent APAs and demonstrated that APA design influences ND diameter and stability through factors such as tandem repeats, sequence composition, and lipid-to-APA ratio.…”

Dual-Targeted Nanodiscs Revealing the Cross-Talk between Osteogenic Differentiation of Mesenchymal Stem Cells and Macrophages

“…26,41,42 The ability of ND to target macrophages was accomplished by DPPS, 43 and the ND assemblies were prepared according to a previously reported method with some modifications. 28 Briefly, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) and 1,2dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) were dissolved in chloroform in a glass tube at a 9:1 molar ratio. Chloroform was dried using a gentle argon gas stream, and the tube was placed in a vacuum desiccator overnight to remove residual solvent.…”

“…In addition, cell-level experiments revealed that KDM6B promoted the differentiation of BMDM into M2. Endogenous and synthetic lipoproteins, such as high-density lipoprotein (HDL) nanodiscs, have emerged as a promising drug delivery system (DDS) for small molecules, peptides, and nucleic acids due to their inherent long half-life in vivo and passive targeting characteristics. , Based on our recently reported work on reprogrammable peptide analogs of apolipoprotein A-I (APA) for tunable nanodisc engineering, we propose a GSK-J4-loaded nanodisc (ND-GSK-J4) targeting macrophages in bone by introducing a bone formation surface-targeting peptide-modified membrane scaffold (SDSSD-APA) and administered it to mice. After administration, it was noted that macrophages in the bone marrow of mice tended to exhibit the M1 type, and bone loss increased.…”

Translational Challenges and Prospective Solutions in the Implementation of Biomimetic Delivery Systems

“…The assembly involves the self-assembly of phospholipids and MSPs, and the correct protein-to-lipid ratio is crucial for ND integrity and function [150]. Factors such as lipid type, MSP variant, and assembly conditions necessitate optimization and significantly impact the assembly complexity and reproducibility [150,151], which are essential for complying with strict pharmaceutical regulations. Minor variations could alter ND properties, affecting their in vivo behavior and therapeutic efficacy, leading to batch variability and translational challenges.…”