Singh Juhi, Raina Rahul, Vinothkumar Kutti R, Anand Ruchi
Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai400076, India.
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bengaluru560065, India.
ACS Chem Biol. 2022 Apr 15;17(4):829-839. doi: 10.1021/acschembio.1c00732. Epub 2022 Mar 22.
Methylation of specific nucleotides is integral for ribosomal biogenesis and also serves as a common mechanism to confer antibiotic resistance by pathogenic bacteria. Here, by determining the high-resolution structure of the 30S-KsgA complex by cryo-electron microscopy, a state was captured, where KsgA juxtaposes between helices h44 and h45 of the 30S ribosome, separating them, thereby enabling remodeling of the surrounded rRNA and allowing the cognate site to enter the methylation pocket. With the structure as a guide, several mutant versions of the ribosomes, where interacting bases in the catalytic helix h45 and surrounding helices h44, h24, and h27, were mutated and evaluated for their methylation efficiency revealing factors that direct the enzyme to its cognate site with high fidelity. The biochemical studies show that the three-dimensional environment of the ribosome enables the interaction of select loop regions in KsgA with the ribosome helices paramount to maintain selectivity.
特定核苷酸的甲基化对于核糖体生物合成至关重要，也是病原菌产生抗生素抗性的常见机制。在此，通过冷冻电子显微镜确定30S-KsgA复合物的高分辨率结构，捕捉到了一种状态，即KsgA并列于30S核糖体的h44和h45螺旋之间，将它们分开，从而使周围的rRNA得以重塑，并使同源位点进入甲基化口袋。以该结构为指导，构建了几种核糖体突变体，其中催化螺旋h45以及周围螺旋h44、h24和h27中的相互作用碱基发生了突变，并对其甲基化效率进行了评估，揭示了指导酶以高保真度靶向其同源位点的因素。生化研究表明，核糖体的三维环境使得KsgA中特定环区域与核糖体螺旋之间能够相互作用，这对于维持选择性至关重要。
