1dfa
CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUPCRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP
Structural highlights
Function[VATA_YEAST] Catalytic subunit of the peripheral V1 complex of vacuolar ATPase. V-ATPase (vacuolar ATPase) is responsible for acidifying a variety of intracellular compartments in eukaryotic cells. It is an electrogenic proton pump that generates a proton motive force of 180 mV, inside positive and acidic, in the vacuolar membrane vesicles. It may participate in maintenance of cytoplasmic Ca(2+) homeostasis. This is a catalytic subunit.[1] PI-SceI is an endonuclease that can cleave at a site present in a VMA1 allele that lacks the derived endonuclease segment of the open reading frame; cleavage at this site only occurs during meiosis and initiates "homing", a genetic event that converts a VMA1 allele lacking VDE into one that contains it.[2] 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[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
|
| ||||||||||||||||||