The antibiotic thiostrepton inhibits a functional transition within protein L11 at the ribosomal GTPase centre

被引:103
作者
Porse, BT
Leviev, I
Mankin, AS
Garrett, RA
机构
[1] Univ Copenhagen, Inst Mol Biol, RNA Regulat Ctr, DK-1307 Copenhagen K, Denmark
[2] Univ Illinois, Ctr Pharmaceut Biotechnol MC870, Chicago, IL 60607 USA
关键词
ribosomal GTPase centre; thiostrepton; drug inhibitory mechanism; ribosomal protein L11; protein footprinting;
D O I
10.1006/jmbi.1997.1541
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A newly identified class of highly thiostrepton-resistant mutants of the archaeon Halobacterium halobium carry a missense mutation at codon 18 within the gene encoding ribosomal protein L11. In the mutant proteins, a proline, conserved in archaea and bacteria, is converted to either serine or threonine. The mutations do not impair either the assembly of the mutant L11 into 70 S ribosomes in vivo or the binding of thiostrepton to ribosomes in vitro. Moreover, the corresponding mutations at proline 22, in a fusion protein of L11 from Escherichia coli with glutathione-S-transferase, did not reduce the binding affinities of the mutated L11 fusion proteins for rRNA or of thiostrepton for the mutant L11-rRNA complexes at rRNA concentrations lower than those prevailing in vivo. Probing the structure of the fusion protein of wild-type L11, from E. coli, using a recently developed protein footprinting technique, demonstrated that a general tightening of the C-terminal domain occurred on rRNA binding, while thiostrepton produced a footprint centred on tyrosine 62 at the junction of the N and C-terminal domains of protein L11 complexed to rRNA. The intensity of this protein footprint was strongly reduced for the mutant L11-rRNA complexes. These results indicate that although, as shown earlier, thiostrepton binds primarily to 23 S rRNA, the drug probably inhibits peptide elongation by impeding a conformational change within protein L11 that is important for the function of the ribosomal GTPase centre. This putative inhibitory mechanism of thiostrepton is critically dependent on proline 18/22. Moreover, the absence of this proline from eukaryotic protein L11 sequences would account for the high thiostrepton resistance of eukaryotic ribosomes. (C) 1998 Academic Press Limited.
引用
收藏
页码:391 / 404
页数:14
相关论文
共 49 条