Wang S, Huber P W, Cui M, Czarnik A W, Mei H Y
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1998 Apr 21;37(16):5549-57. doi: 10.1021/bi972808a.
Neomycin inhibits the binding of Tat-derived peptides to the trans-activating region (TAR) of HIV-1 RNA. Kinetic studies reveal that neomycin acts as a noncompetitive inhibitor that can bind to the Tat-TAR complex and increase the rate constant (koff) for dissociation of the peptide from the RNA. Neomycin effects a conformational change in the structure of TAR that can be detected by circular dichroism spectroscopy. The increase in ellipticity measured at 265 nm upon binding of the aminoglycoside is opposite to the decrease seen when Tat peptides bind to the RNA. Thus, the structural transition induced by neomycin is apparently incompatible with the binding of Tat and underlies the inhibitory action of the antibiotic. The binding site for neomycin on TAR was identified in ribonuclease protection experiments and is located in the stem immediately below the three-nucleotide bulge that serves as the primary identity element for Tat. Apparent protection of residues in the bulge by neomycin may represent additional contacts to the aminoglycoside, but more likely result from changes in the structure of this region when the ligand binds to the RNA. Binding assays using variants of TAR in which inosine residues were substituted for guanosine residues support the results from the ribonuclease protection experiments. Inosine substitutions in the lower stem, but not the upper stem, decrease the binding constant for neomycin by approximately 100-fold. Neither of these variants affected the binding affinity of Tat peptide. In addition, these latter experiments suggest that the aminoglycoside may be located in the minor groove of the stem. This mode of association may be a critical aspect of neomycin's ability to bind to the Tat-TAR complex and could serve as a guide for the design of other drugs that bind to specific RNA targets as noncompetitive inhibitors.
新霉素可抑制Tat衍生肽与HIV-1 RNA反式激活区域（TAR）的结合。动力学研究表明，新霉素作为一种非竞争性抑制剂，可与Tat-TAR复合物结合，并增加肽从RNA上解离的速率常数（koff）。新霉素可使TAR结构发生构象变化，这种变化可用圆二色光谱法检测到。氨基糖苷类药物结合后在265 nm处测得的椭圆率增加与Tat肽与RNA结合时观察到的减少相反。因此，新霉素诱导的结构转变显然与Tat的结合不相容，这是该抗生素抑制作用的基础。在核糖核酸酶保护实验中确定了新霉素在TAR上的结合位点，它位于紧邻作为Tat主要识别元件的三核苷酸凸起下方的茎区。新霉素对凸起处残基的明显保护可能代表与氨基糖苷类药物的额外接触，但更可能是由于配体与RNA结合时该区域结构的变化。使用用肌苷残基替代鸟苷残基的TAR变体进行的结合试验支持了核糖核酸酶保护实验的结果。下茎区而非上茎区的肌苷替代使新霉素的结合常数降低约100倍。这些变体均未影响Tat肽的结合亲和力。此外，这些后续实验表明氨基糖苷类药物可能位于茎区的小沟中。这种结合方式可能是新霉素与Tat-TAR复合物结合能力的关键方面，并可为设计其他作为非竞争性抑制剂与特定RNA靶点结合的药物提供指导。
