Hydrophobic cavity in C-terminus is essential for hTNF-alpha trimer conformation

摘要

A variety of tumour necrosis factor alpha (TNF-alpha) derivatives have been bioengineered to improve antitumour activity and reduce toxicity. The expression of TNF-alpha in Escherichia coli usually yields a mixture of homotrimers and monomers; however, only the trimer shows antitumour activity. TNF-alpha D10, a bioengineered hTNF-alpha derivative, demonstrated 10-fold higher cytotoxicity against tumour cells compared to hINF-alpha, 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-alpha D10. 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-alpha 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-alpha trimer production. (C) 2011 Elsevier Masson SAS. All rights reserved.