title: Hydrophobic cavity in C-terminus is essential for hTNF-α trimer conformation.
abstract: A variety of tumour necrosis factor α (TNF-α) derivatives have been bioengineered to improve antitumour activity and reduce toxicity. The expression of TNF-α in Escherichia coli usually yields a mixture of homotrimers and monomers; however, only the trimer shows antitumour activity. TNF-αD10, a bioengineered hTNF-α derivative, demonstrated 10-fold higher cytotoxicity against tumour cells compared to hTNF-α, but the trimer to monomer ratio was 58:42. In the present study, we investigated the structural differences between the trimer and the monomer of TNF-αD10. We found that the chemical shifts of the C-terminal Trp(114) in the trimer were significantly different from those in the monomer and that the replacement of Trp(114) with different amino acids remarkably reduced the trimer production. Further analysis of the publicly available X-ray crystallographic data for trimeric and monomeric hTNF-α revealed that the conformation of the U-shaped region formed by the fragment Cys(101)-Trp(114) was different between the two forms: a hydrophilic cavity in the monomer and a hydrophobic cavity in the trimer. These findings suggested the potential approaches of molecular and structural modification for future improvement of hTNF-α trimer production.
authors.affiliation: State Key Laboratory of Genetic Engineering, Department of Biochemistry, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.forename: Linsen
authors.initials: L
authors.lastname: Dai
authors.forename: Zhaojing
authors.initials: Z
authors.lastname: Hao
authors.forename: Weida
authors.initials: W
authors.lastname: Huang
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D006801:Humans; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha