Singh Avinash, Kumar Ashok, Gautam Lovely, Sharma Pradeep, Sinha Mau, Bhushan Asha, Kaur Punit, Sharma Sujata, Arora Ashish, Singh Tej P
*Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, 110029 Delhi, India.
†Molecular and Structural Biology Division, Central Drug Research Institute, 226031 Lucknow, India.
Biochem J. 2014 Nov 1;463(3):329-37. doi: 10.1042/BJ20140631.
During the course of protein synthesis in the cell, the translation process is often terminated due to various reasons. As a result, peptidyl-tRNA molecules are released which are toxic to the cell as well reducing the availability of free amino acid and tRNA molecules for the required protein synthesis in the cell. Such a situation is corrected by an enzyme, Pth (peptidyl-tRNA hydrolase), which catalyses the release of free tRNA and peptide moieties from peptidyl-tRNAs. This means that the active Pth is essential for the survival of bacteria. In order to design inhibitors of PaPth (Pth from Pseudomonas aeruginosa), we determined the structures of PaPth in its native and bound states with compounds amino acylate-tRNA analogue and 5-azacytidine. The structure determination of the native protein revealed that the substrate-binding site was partially occupied by Glu161 from the neigh-bouring molecule. The structure of PaPth indicated that the substrate-binding site can be broadly divided into three distinct subsites. The structures of the two complexes showed that the amino acylate-tRNA analogue filled three subsites, whereas 5-azacytidine filled two subsites. The common sugar and the base moieties of the two compounds occupied identical positions in the cleft. Using surface plasmon resonance, the dissociation constants for the amino acylate-tRNA analogue and 5-azacytidine were found to be 3.53×10-8 M and 5.82×10-8 M respectively.
在细胞内蛋白质合成过程中，翻译过程常常因各种原因而终止。结果，肽基 - tRNA分子被释放出来，这些分子对细胞有毒性，同时也减少了细胞内用于所需蛋白质合成的游离氨基酸和tRNA分子的可用性。一种名为Pth（肽基 - tRNA水解酶）的酶可纠正这种情况，它催化从肽基 - tRNA中释放出游离的tRNA和肽部分。这意味着活性Pth对细菌的存活至关重要。为了设计铜绿假单胞菌Pth（PaPth）的抑制剂，我们测定了PaPth在其天然状态以及与氨基酰化 - tRNA类似物和5 - 氮杂胞苷结合状态下的结构。天然蛋白质的结构测定表明，底物结合位点被来自相邻分子的Glu161部分占据。PaPth的结构表明，底物结合位点可大致分为三个不同的亚位点。两种复合物的结构显示，氨基酰化 - tRNA类似物占据了三个亚位点，而5 - 氮杂胞苷占据了两个亚位点。这两种化合物的共同糖基和碱基部分在裂隙中占据相同位置。使用表面等离子体共振技术，发现氨基酰化 - tRNA类似物和5 - 氮杂胞苷的解离常数分别为3.53×10⁻⁸ M和5.82×10⁻⁸ M。
