3pvi
From Proteopedia
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D34G MUTANT OF PVUII ENDONUCLEASE COMPLEXED WITH COGNATE DNA SHOWS THAT ASP34 IS DIRECTLY INVOLVED IN DNA RECOGNITION AND INDIRECTLY INVOLVED IN CATALYSIS
Overview
The PvuII restriction endonuclease is a homodimer that recognizes and, cleaves the DNA sequence 5'-CAGCTG-3' in double-stranded DNA, and the, structure of this enzyme has been reported. In the wild-type enzyme, Asp34, interacts with the internal guanine of the recognition sequence on the, minor groove side. The Asp34 codon was altered to specify Gly (D34G), and, in vitro studies have revealed that the D34G protein has lost binding, specificity for the central G.C base-pairs, and that it cuts the canonical, sequence with 10(-4)-fold reduced activity as compared to the wild-type, enzyme. We have now determined the structure at 1.59 A resolution of the, D34G PvuII endonuclease complexed with a 12 bp duplex deoxyoligonucleotide, containing the cognate sequence. The D34G alteration results in several, structural changes relative to wild-type protein/DNA complexes. First, the, sugar moiety of the internal guanine changes from a C2'-endo to C3'-endo, pucker while that of the 3' guanine changes from C3'-endo to C2'-endo, pucker. Second, the axial rise between the internal G.C base-pairs is, reduced while that between the G.C and flanking base-pairs is expanded., Third, two distinct monomeric active sites are observed that we refer to, as being "primed" and "unprimed" for phosphodiester bond cleavage. The, primed and unprimed sites differ in the conformation of the Asp58, side-chain, and in the absence from unprimed sites of four networked water, molecules. These water molecules, present in the primed site, have been, implicated in the catalytic mechanism of this and other endonucleases;, some of them can be replaced by the Mg2+ necessary for cleavage. Taken, together, these structural changes imply that the Asp34 side-chains from, the two subunits maintain a distinct conformation of its DNA substrate, properly situating the target backbone phosphates and indirectly, manipulating the active sites. This provides some insight into how, recognition of the specific DNA sequence is linked to catalysis by the, highly specific restriction endonucleases, and reveals one way in which, the structural conformation of the DNA is modulated coordinately with that, of the PvuII protein.
About this Structure
3PVI is a Single protein structure of sequence from Proteus vulgaris. Full crystallographic information is available from OCA.
Reference
Asp34 of PvuII endonuclease is directly involved in DNA minor groove recognition and indirectly involved in catalysis., Horton JR, Nastri HG, Riggs PD, Cheng X, J Mol Biol. 1998 Dec 18;284(5):1491-504. PMID:9878366
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