The molecular basis for A-site mutations conferring aminoglycoside resistance:: Relationship between ribosomal susceptibility and X-ray crystal structures
The molecular basis for A-site mutations conferring aminoglycoside resistance:: Relationship between ribosomal susceptibility and X-ray crystal structures
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DOI：
10.1002/cbic.200300657
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发表时间：
2003-10-06
期刊：
影响因子：
3.2
通讯作者：
Westhof, E
中科院分区：
文献类型：
--
作者：
Pfister, P;Hobbie, S;Westhof, E
氨基糖苷类抗生素以16S核糖体RNA(RRNA)细菌A位为靶点，并诱导遗传密码的误读。核糖体A位点的点突变可能导致对氨基糖苷类抗生素的耐药性。通过测定最低抑菌浓度，研究细菌突变(通过定点突变引入)对体内核糖体药物敏感性的影响。为了在分子水平上确定不同耐药表型的来源，将体内结果与先前发表的与含有最小A位点的寡核苷酸结合的巴洛霉素、妥布霉素和遗传素的晶体结构进行了比较。有两个区域似乎对A位点的结合至关重要：1408位的单腺嘌呤残基和非Watson-Crick U1406 U1495对。这些位置的突变的影响受环I上6‘位取代基(羟基或氨基)的性质、抗生素上的正电荷数量以及环I和环III(4、5或4、6)之间的连接所调节。特别是，分析表明：1)C1409-G1491到A1409-U1491的多态(在15%的细菌中观察到)与耐药性无关，这表明它不影响环I在残基1491上的堆积，2)A1408G突变表现出对6‘-NH3+氨基糖苷类药物的高水平抗性很可能是由于环I不能与G1408形成伪碱基对而阻止其插入A位螺旋，3)形成U1406 U1495对的尿嘧啶残基对胞嘧啶或腺嘌呤残基的突变大多产生低到中等水平的耐药性，而U1406C/U1495A双突变产生高水平的耐药性(新霉素除外)，这表明氨基糖苷类与野生型A位点的结合及其功能结果强烈依赖于U1406 U1495对的特定几何结构。体内观察到的抗性表型与分子水平上描述的相互作用之间的关系决定了X射线结晶学研究中观察到的不同结构相互作用的生物学重要性。
Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site and induce misreading of the genetic code. Point mutations of the ribosomal A site may confer resistance to aminoglycoside antibiotics. The influence of bacterial mutations (introduced by site-directed mutagenesis) on ribosomal drug susceptibility was investigated in vivo by determination of minimal inhibitory concentrations. To determine the origin of the various resistance phenotypes at a molecular level, the in vivo results were compared with the previously published crystal structures of paromomycin, tobramycin,and geneticin bound to oligonucleotides containing the minimal A site. Two regions appear crucial for binding in the A site: the single adenine residue at position 1408 and the non-Watson-Crick U1406 U1495 pair. The effects of mutations at those position are modulated by the nature of the substituent at position 6' (either hydroxy or ammonium group) on ring I, by the number of positive charges on the antibiotic, and by the linkage between rings I and III (either 4, 5 or 4,6). In particular, the analysis demonstrates: 1) that the C1409-G1491 to A1409 - U1491 polymorphism (observed in 15% of bacteria) is not associated with resistance, which indicates that it does not affect the stacking of ring I on residue 1491, 2) that the high-level resistance to 6'-NH3+ aminoglycosides exhibited by the A1408G mutation most probably results from the inability of ring I forming a pseudo base pair with G1408 which prevents its insertion inside the A site helix, and 3) that mutations of the uracil residues forming the U1406 U1495 pair either to cytosine or to adenine residues mostly confer low to moderate levels of drug resistance whereas the U1406C/U1495A double mutation confers high-level resistance (except for neomycin) which suggests that aminoglycoside binding to the wild-type A site and its functional consequences strongly depend on a particular geometry of the U1406 U1495 pair. The relationships between the resistance phenotypes observed in vivo and the interactions described at the molecular level define the biological importance of the different structural interactions observed by X-ray crystallography studies.