Mutational analysis of vaccinia virus mRNA cap (guanine-N7) methyltransferase reveals essential contributions of the N-terminal peptide that closes over the active site

被引:9
作者
Zheng, Sushuang [1 ]
Shuman, Stewart [1 ]
机构
[1] Sloan Kettering Inst, Program Mol Biol, New York, NY 10065 USA
关键词
S-adenosylmethionine; mRNA capping; poxvirus; sulfonium;
D O I
10.1261/rna.1201308
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
RNA guanine-N7 methyltransferase catalyzes the third step of eukaryal mRNA capping, the transfer of a methyl group from AdoMet to GpppRNA to form m 7 GpppRNA. Mutational and crystallographic analyses of cellular and poxvirus cap methyltransferases have yielded a coherent picture of a conserved active site and determinants of substrate specificity. Models of the Michaelis complex suggest a direct in-line mechanism of methyl transfer. Because no protein contacts to the guanine-N7 nucleophile, the AdoMet methyl carbon (C epsilon) or the AdoHcy sulfur (S delta) leaving group were observed in ligand-bound structures of cellular cap methyltransferase, it was initially thought that the enzyme facilitates catalysis by optimizing proximity and geometry of the donor and acceptor. However, the structure of AdoHcy-bound vaccinia virus cap methyltransferase revealed the presence of an N-terminal "lid peptide" that closes over the active site and makes multiple contacts with the substrates, including the AdoMet sulfonium. This segment is disordered in the vaccinia apoenzyme and is not visible in the available structures of cellular cap methyltransferase. Here, we conducted a mutational analysis of the vaccinia virus lid peptide ((545)DKFRLNPEVSYFTNKRTRG(563)) entailing in vivo and in vitro readouts of the effects of alanine and conservative substitutions. We thereby identified essential functional groups that interact with the AdoMet sulfonium (Tyr555, Phe556), the AdoMet adenine (Asn550), and the cap triphosphate bridge (Arg560, Arg562). The results suggest that van der Waals contacts of Tyr555 and Phe556 to the AdoMet Sd and Ce atoms, and the electron-rich environment around the sulfonium, serve to stabilize the transition state of the transmethylation reaction.
引用
收藏
页码:2297 / 2304
页数:8
相关论文
共 24 条
[1]   mRNA: guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships [J].
Bujnicki, Janusz M. ;
Feder, Marcin ;
Radlinska, Monika ;
Rychlewski, Leszek .
BMC BIOINFORMATICS, 2001, 2 (1)
[2]  
CONG PJ, 1992, J BIOL CHEM, V267, P16424
[3]   Structural insights into the mechanism and evolution of the vaccinia virus mRNA cap N7 methyl-transferase [J].
De la Pena, Marcos ;
Kyrieleis, Otto J. P. ;
Cusack, Stephen .
EMBO JOURNAL, 2007, 26 (23) :4913-4925
[4]   Structure and mechanism of mRNA cap (guanine-N7) methyltransferase [J].
Fabrega, C ;
Hausmann, S ;
Shen, V ;
Shuman, S ;
Lima, CD .
MOLECULAR CELL, 2004, 13 (01) :77-89
[5]   Encephalitozoon cuniculi mRNA cap (guanine N-7) methyltransferase [J].
Hausmann, S ;
Zheng, SS ;
Fabrega, C ;
Schneller, SW ;
Lima, CD ;
Shuman, S .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2005, 280 (21) :20404-20412
[6]  
HIGMAN MA, 1994, J BIOL CHEM, V269, P14974
[7]  
HIGMAN MA, 1992, J BIOL CHEM, V267, P16430
[8]   THE D1 AND D12 SUBUNITS ARE BOTH ESSENTIAL FOR THE TRANSCRIPTION TERMINATION FACTOR ACTIVITY OF VACCINIA VIRUS CAPPING ENZYME [J].
LUO, Y ;
MAO, XD ;
DENG, L ;
CONG, PJ ;
SHUMAN, S .
JOURNAL OF VIROLOGY, 1995, 69 (06) :3852-3856
[9]  
MAO XD, 1994, J BIOL CHEM, V269, P24472
[10]   Vaccinia virus mRNA (guanine-7-)methyltransferase: Mutational effects on cap methylation and AdoHcy-dependent photo-cross-linking of the cap to the methyl acceptor site [J].
Mao, XD ;
Shuman, S .
BIOCHEMISTRY, 1996, 35 (21) :6900-6910