Pfister Peter, Hobbie Sven, Vicens Quentin, Böttger Erik C, Westhof Eric
Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30/32, 8028 Zürich, Switzerland.
Chembiochem. 2003 Oct 6;4(10):1078-88. doi: 10.1002/cbic.200300657.
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 positions 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.
氨基糖苷类抗生素作用于细菌16S核糖体RNA（rRNA）的A位点，诱导遗传密码错读。核糖体A位点的点突变可能导致对氨基糖苷类抗生素产生抗性。通过测定最低抑菌浓度，在体内研究了（通过定点诱变引入的）细菌突变对核糖体药物敏感性的影响。为了在分子水平上确定各种抗性表型的起源，将体内实验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龙霉素、妥布霉素和庆大霉素的晶体结构进行了比较。A位点中有两个区域对于结合至关重要：位置1408处的单个腺嘌呤残基以及非沃森-克里克U1406.U1495碱基对。这些位置突变的影响受到I环上6'位（羟基或铵基）取代基的性质、抗生素上正电荷的数量以及I环与III环之间的连接（4,5或4,6）的调节。具体而言，分析表明：1）C1409-G1491到A1490-U1491的多态性（在15%的细菌中观察到）与抗性无关，这表明它不影响I环在残基1491上的堆积；2）A1408G突变对6'-NH3+氨基糖苷类抗生素表现出的高水平抗性很可能是由于I环无法与G1408形成假碱基对，从而阻止其插入A位点螺旋内部；3）形成U1406.U1495碱基对的尿嘧啶残基突变为胞嘧啶或腺嘌呤残基大多导致低至中等水平的耐药性，而U1406C/U1495A双突变则导致高水平抗性（新霉素除外）。这表明氨基糖苷类抗生素与野生型A位点的结合及其功能后果强烈依赖于U1406.U1495碱基对的特定几何结构。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，确定了X射线晶体学研究中观察到的不同结构相互作用的生物学重要性。
