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CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUPCRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP
Structural highlights
Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedThe PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure. Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.,Hu D, Crist M, Duan X, Quiocho FA, Gimble FS J Biol Chem. 2000 Jan 28;275(4):2705-12. PMID:10644733[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences |
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