ABSTRACT
N(2)-methylguanosine is one of the most universal modified nucleosides required for proper function in transfer RNA (tRNA) molecules. In archaeal tRNA species, a specific S-adenosyl-L-methionine (SAM)-dependent tRNA methyltransferase (MTase), aTrm11, catalyzes formation of N(2)-methylguanosine and N(2),N(2)-dimethylguanosine at position 10. Here, we report the first X-ray crystal structures of aTrm11 from Thermococcus kodakarensis (Tko), of the apo-form, and of its complex with SAM. The structures show that TkoTrm11 consists of three domains: an N-terminal ferredoxinlike domain (NFLD), THUMP domain and Rossmann-fold MTase (RFM) domain. A linker region connects the THUMP-NFLD and RFM domains. One SAM molecule is bound in the pocket of the RFM domain, suggesting that TkoTrm11 uses a catalytic mechanism similar to that of other tRNA MTases containing an RFM domain. Furthermore, the conformation of NFLD and THUMP domains in TkoTrm11 resembles that of other tRNA-modifying enzymes specifically recognizing the tRNA acceptor stem. Our docking model of TkoTrm11-SAM in complex with tRNA, combined with biochemical analyses and pre-existing evidence, provides insights into the substrate tRNA recognition mechanism: The THUMP domain recognizes a 3'-ACCA end, and the linker region and RFM domain recognize the T-stem, acceptor stem and V-loop of tRNA, thereby causing TkoTrm11 to specifically identify its methylation site.
Subject(s)
DNA Methylation/genetics , RNA, Transfer/genetics , Thermococcus/chemistry , tRNA Methyltransferases/chemistry , Amino Acid Sequence/genetics , Binding Sites , Crystallography, X-Ray , Guanosine/analogs & derivatives , Guanosine/chemistry , Guanosine/metabolism , Molecular Docking Simulation , RNA, Transfer/chemistry , RNA-Binding Motifs/genetics , S-Adenosylmethionine/chemistry , Sequence Alignment , Thermococcus/enzymology , tRNA Methyltransferases/metabolismABSTRACT
Case 1: A 69-year-old man was diagnosed with rectal cancer and liver metastasis. After low anterior resection, mFOLFOX6 plus cetuximab therapy was started for resection of the liver metastasis. However, he had to forgo liver resection because he developed acute exacerbation of interstitial pneumonitis (IP) after 6 courses of chemotherapy. Despite beginning the second-line treatment with mFOLFOX6 plus bevacizumab, he died in June 2012. Case 2: A 71-year-old man had undergone sigmoidectomy for sigmoid colon cancer in 2005, and right lower lobe partial resection for metastatic lung cancer in 2006. Although radiofrequency ablation or transcatheter arterial chemoembolization had been performed for multiple liver metastases several times since 2007, his multiple liver metastases were uncontrollable. Therefore, FOLFOX4 therapy was started in 2010, and mFOLFOX6 plus cetuximab therapy was substituted for FOLFOX4 therapy in 2011. The patient died in March 2012 due to the rapid development of IP, and thus, it appears that IP was the cause of death in both patients. The general condition, including pulmonary function, of patients at risk of IP must be checked before starting cetuximab therapy for metastatic colorectal cancer.
