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PDBsum entry 1az3

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Endonuclease PDB id
1az3
Jmol
Contents
Protein chains
186 a.a.
215 a.a.
Waters ×38

References listed in PDB file
Key reference
Title Conformational transitions and structural deformability of ecorv endonuclease revealed by crystallographic analysis.
Authors J.J.Perona, A.M.Martin.
Ref. J Mol Biol, 1997, 273, 207-225. [DOI no: 10.1006/jmbi.1997.1315]
PubMed id 9367757
Abstract
The structures of wild-type and mutant forms of the unliganded EcoRV endonuclease dimer have been determined at 2.4 A resolution in a new crystal lattice. Comparison of these structures with that of the free enzyme determined with different packing constraints shows that the conformations of the domain interfaces are not conserved between crystal forms. The unliganded enzyme and the enzyme-DNA complex delineate two distinct quaternary states separated by a 25 degrees intersubunit rotation, but considerable conformational heterogeneity, of the order of 10 degrees domain rotations, exists within each of these states. Comparison of the free enzyme structure between the two crystal forms further reveals that the C-terminal 28 amino acid residues are disordered and undergo an extensive local folding transition upon DNA binding. Introduction of the mutation T93A at the DNA-binding cleft causes large-scale effects on the protein conformation. Structural changes in the mutated unliganded enzyme propagate some 20 to 25 A to the dimerization interface and lead to a rearrangement of monomer subunits. Comparative analysis of these structures, a new structure of the enzyme cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino acid residues in facilitating the intersubunit motions and local folding transitions. In particular, the T93A structure reveals a pathway through the protein, by which DNA-binding may cause the domain movements required for proper alignment of catalytic groups. The key active-site residue Glu45 is located on a flexible helix inside this pathway, and this provides a direct means by which essential catalytic functions are coupled to the protein conformational change. It appears that indirect perturbation of the Glu45 conformation via an altered quaternary structure may be a contributing factor to the decreased catalytic efficiency of T93A, and this mechanism may also explain the diminished activities of other active site variants of EcoRV.
Figure 7.
Figure 7. (a) Ribbon representation of the crystal struc- ture of the EcoRV-DNA complex showing the location of residue Thr93 in the DNA binding cleft, and its pos- ition with respect to helix B and the dimer interface region (DIM). (b) Stereo view of a superposition of WTUCB (yellow) and T93A (orange) using polypeptide backbone atoms within one of the DNA-binding domains, as defined by difference-distance calculations. A steric clash between Phe47 of T93A (orange) and Val20 of WTUCB (yellow) shows the origin of the domain reorientation in the mutant.
Figure 8.
Figure 8. (a) Stereo view of the active site of the ternary complex of EcoRV cocrystallized with cognate DNA and Ca 2+ . The position of the leaving 30 oxygen atom of the scissile bond is indicated. In this conformation, in-line attack of the hydroxide ion to form the expected trigonal bipyramid in the transition state must be from the face opposite the position of the carboxylate groups. The calcium ion (#283 CL) binds between Asp90, Asp74 and the scissile phos- phate groups. Helix B is at the left. (b) Detailed view of the ligation of the calcium ion in subunit I of the EcoRV- DNA-Ca 2+ complex, including distances (in Å ) as determined from the final refined coordinate set. The scissile phos- phate group is shown at the top. The ligation of Ca 2+ in subunit II is very similar, except that electron density for one apical water molecule is not apparent in electron density maps, and the two oxygen atoms of Asp90 are located in the equatorial plane at distances of 2.53 Å and 2.46 Å from the calcium ion.
The above figures are reprinted by permission from Elsevier: J Mol Biol (1997, 273, 207-225) copyright 1997.
Secondary reference #1
Title The crystal structure of ecorv endonuclease and of its complexes with cognate and non-Cognate DNA fragments.
Authors F.K.Winkler, D.W.Banner, C.Oefner, D.Tsernoglou, R.S.Brown, S.P.Heathman, R.K.Bryan, P.D.Martin, K.Petratos, K.S.Wilson.
Ref. EMBO J, 1993, 12, 1781-1795.
PubMed id 8491171
Abstract
Secondary reference #2
Title Structure and function of restriction endonucleases
Author F.K.Winkler.
Ref. curr opin struct biol, 1992, 2, 93.
PROCHECK
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