Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies

Abstract

Objective: During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF–IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied.

Research design and methods: Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics.

Results: Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 – 111) superficially interacts with similar TLR4/MD-2 (K_D = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 – 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS.

Conclusions: ProTα via its C-terminal peptide (aa 91 – 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

Keywords:

• gravimetric analysis
• lipopolysaccharide
• molecular dynamics simulation
• prothymosin alpha
• Toll-like receptor 4

Acknowledgment

Both authors equally contributed to this study.

Declaration of interest

This paper is part of a supplemental issue, sponsored by SciClone. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science and in part by Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Hiroshi Ueda has received Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

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