title: Structure of a hyperthermophilic archaeal homing endonuclease, I-Tsp061I: contribution of cross-domain polar networks to thermostability.
abstract: 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 A 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 alpha-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.
authors.affiliation: Structural Biophysics Laboratory, RIKEN SPring-8 Center, Harima Institute, Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Takahito
authors.initials: T
authors.lastname: Imagawa
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Shirai
authors.forename: Takashi
authors.initials: T
authors.lastname: Itoh
authors.forename: Yoshihiko
authors.initials: Y
authors.lastname: Sako
authors.forename: Masashi
authors.initials: M
authors.lastname: Miyano
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Sakuraba
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Ohshima
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004720:Endonucleases / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D041542:Thermoproteus / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D004720:Endonucleases