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'''Co-crystal structure of E.coli RNase HI active site mutant (E48A/D134N*) with Mn2+''' | '''Co-crystal structure of E.coli RNase HI active site mutant (E48A/D134N*) with Mn2+''' | ||
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[[Category: Tsunaka, Y.]] | [[Category: Tsunaka, Y.]] | ||
[[Category: Yamagata, Y.]] | [[Category: Yamagata, Y.]] | ||
[[Category: | [[Category: Active-site mutant]] | ||
[[Category: | [[Category: Co-crystal structure with mn2+]] | ||
[[Category: | [[Category: Metal fluctuation model]] | ||
[[Category: | [[Category: Rnase h]] | ||
[[Category: | [[Category: X-ray crystallography]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sat May 3 14:05:02 2008'' | |||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | |||
Revision as of 14:05, 3 May 2008
Co-crystal structure of E.coli RNase HI active site mutant (E48A/D134N*) with Mn2+
OverviewOverview
Escherichia coli RNase HI has two Mn(2+)-binding sites. Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134. Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively. However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity. In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined. In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala. In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala. In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn. All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions. These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket. These positions are different from one another, but none of them is similar to site 1. The temperature factors of these manganese ions were considerably larger than those of the surrounding residues. These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets. We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model). The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion.
About this StructureAbout this Structure
1WSG is a Single protein structure of sequence from Escherichia coli. Full crystallographic information is available from OCA.
ReferenceReference
Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography., Tsunaka Y, Takano K, Matsumura H, Yamagata Y, Kanaya S, J Mol Biol. 2005 Feb 4;345(5):1171-83. Epub 2004 Dec 16. PMID:15644213 Page seeded by OCA on Sat May 3 14:05:02 2008