Structure of a Hyperthermophilic Archaeal Homing Endonuclease, I-Tsp061I: Contribution of Cross-domain Polar Networks to Thermostability

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• 作者：Hitoshi Nakayama ; Tatsuro Shimamura ; Takahito Imagawa ; Nobuaki Shirai ; Takashi Itoh ; Yoshihiko Sako ; Masashi Miyano ; Haruhiko Sakuraba ; Toshihisa Ohshima ; Norimichi Nomura ; et al.
• 关键词：LAGLIDADG-type homing endonuclease ; hyperthermostability ; crystal structure ; mutagenic study ; cross-domain polar network
• 刊名：Journal of Molecular Biology
• 出版年：2007
• 期刊代码：102_00222836
• 类别：imm
• 出版时间：12 January 2007
• 卷：365
• 期：2
• 页码：362-378
• 文件大小：2182 K

摘要

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 Å resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed α-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.