Conserved residues that modulate protein trans-splicing of Npu DnaE split intein.
The first crystal trans-structure of a naturally occurring split intein has been determined for the Npu (Nostoc punctiforme PCC73102) DnaE split intein. Guided by this structure, the residues NArg50 and CSer35, well conserved in DnaE split inteins, are identified to be critical in the trans-splicing of Npu DnaE split intein. An in vitro splicing assay demonstrates that NArg50 and CSer35 play synergistic roles in modulating its intein activity. The C-terminal CAsn36 exhibits two orientations of its side chain and interacts with both NArg50 and CSer35 through hydrogen bonding. These interactions likely facilitate the cyclization of asparagine in the course of protein splicing. The mutation of either residue reduces intein activity, and correlates with the low activity of the Ssp (Cyanobacterium synechocystis sp. strain PCC6803) DnaE split intein. On the other hand, NArg50 also forms a hydrogen bond with the highly conserved F-block CAsp17, thus influencing the N-S acyl shift during N-terminal cleavage. Sequence alignments show that residues NArg50 and CSer35 are rather conserved in those split inteins that lack a penultimate histidine residue. The conserved non-catalytic residues of split inteins modulate the efficiency of protein trans-splicing by hydrogen-bond interactions with the catalytic residues at the splice junction.
*CAS Key Laboratory of Soft Matter Chemistry, CAS High Magnetic Field Laboratory, Department of Chemistry, University of Science and Technology of China, 230026 Hefei, Anhui, China.
Qin
Q
Wu
†Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.
Zengqiang
Z
Gao
†Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.
Yong
Y
Wei
*CAS Key Laboratory of Soft Matter Chemistry, CAS High Magnetic Field Laboratory, Department of Chemistry, University of Science and Technology of China, 230026 Hefei, Anhui, China.
Guolin
G
Ma
‡Department of Chemistry, Huangshan University, Huangshan, Anhui 245041, China.
Yuchuan
Y
Zheng
†Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China.
Yuhui
Y
Dong
*CAS Key Laboratory of Soft Matter Chemistry, CAS High Magnetic Field Laboratory, Department of Chemistry, University of Science and Technology of China, 230026 Hefei, Anhui, China.
Yangzhong
Y
Liu
D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D047668:Inteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D046937:Nostoc / Q000737:chemistry* / Q000201:enzymology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D046939:Synechocystis / Q000737:chemistry / Q000201:enzymology; D020040:Trans-Splicing
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D012694:Serine; D001120:Arginine; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III