NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein.
NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein.
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DOI：
10.1016/j.jmb.2012.04.029
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发表时间：
2012-08-03
影响因子：
5.6
通讯作者：
Iwai, Hideo
中科院分区：
文献类型：
--
作者：
Oeemig, Jesper S.;Zhou, Dongwen;Kajander, Tommi;Wlodawer, Alexander;Iwai, Hideo
In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the exteinthat follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein fromPyrococcus horikoshii. The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of the less robust splicing variants by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offers a promising way of improving their properties as better tools for protein engineering.
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