Inducing rigid local structure around the zinc-binding region by hydrophobic interactions enhances the homotrimerization and apoptotic activity of zinc-free TRAIL.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), existing as homotrimer in solution, contains a unique zinc-binding site coordinated by three Cys230 residues at the tip of trimeric interface. TRAIL mutant with replacements of Cys230 with Ala (TRAIL(C230A)) negligibly formed trimeric structure and showed no apoptotic activity. Here, to elucidate the relationship between the trimeric stability and the apoptotic activity of TRAIL(C230A), we rationally designed mutations to induce homotrimerization of TRAIL(C230A) by substituting for the three residues involved in hydrogen bonding (Tyr183 and Tyr243) and putative repulsive electrostatic (Arg227) interactions at the buried trimeric interface into hydrophobic residues, like Y183F, Y243F, and R227I. The TRAIL(C230A)-derived mutants exhibited enhanced homotrimerization, but only the mutants containing R227I exhibited significant apoptosis-inducing activity in cancer cells. These results, together with the induction of rigid local structure around the zinc-binding region by R227I in TRAIL(C230A), suggest that ordered, rigid structure around the zinc-binding region is critical for the homotrimerization and apoptotic activity of TRAIL.
Lee
Hyun-Wook
HW
Department of Molecular Science and Technology, Ajou University, San5, Wonchon-dong, Yeongtong-gu, Suwon 443-749, Republic of Korea.
Kim
Tae-In
TI
Chan
Khon Huynh
KH
Kwon
Myung-Hee
MH
Kim
Jeong-Sun
JS
Jin
Moonsoo
M
Kim
Yong-Sung
YS
D017209:Apoptosis; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D015032:Zinc / Q000737:chemistry*
D053221:TNF-Related Apoptosis-Inducing Ligand; D014364:Tryptophan; D015032:Zinc; D003545:Cysteine