title: 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.
  
  
  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 (KD = 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.
authors.affiliation: Nagasaki University Graduate School of Biomedical Sciences, Department of Pharmacology and Therapeutic Innovation , 1-14 Bunkyo-machi, Nagasaki 852-8521 , Japan +81 95 819 2421; +81 95 819 2420; ueda@nagasaki-u.ac.jp.
authors.forename: Olaposi
authors.initials: O
authors.lastname: Omotuyi
authors.forename: Hayato
authors.initials: H
authors.lastname: Matsunaga
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002623:Chemistry Techniques, Analytical; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000737:chemistry / Q000378:metabolism; D015850:Interleukin-6 / Q000737:chemistry / Q000378:metabolism; D051218:Lymphocyte Antigen 96 / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D013947:Thymosin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D015850:Interleukin-6; C495377:LY96 protein, human; D051218:Lymphocyte Antigen 96; D011498:Protein Precursors; D017472:Receptors, Interleukin-1; D051197:Toll-Like Receptor 4; C040782:prothymosin alpha; D013947:Thymosin
keywords: Toll-like receptor 4; gravimetric analysis; lipopolysaccharide; molecular dynamics simulation; prothymosin alpha