CONFORMATIONAL TRANSITIONS AND STRUCTURAL DEFORMABILITY OF ECORV ENDONUCLEASE REVEALED BY CRYSTALLOGRAPHIC ANALYSIS

The structures of wild-type and mutant forms of the unliganded EcoRV e ndonuclease dimer have been determined at 2.4 Angstrom resolution in a new crystal lattice. Comparison of these structures with that of the free enzyme determined with different packing constraints shows that t he conformations of the domain interfaces are not conserved between cr ystal forms. The unliganded enzyme and the enzyme-DNA complex delineat e two distinct quaternary states separated by a 25 degrees intersubuni t rotation, but considerable conformational heterogeneity, of the orde r 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 disorde red and undergo an extensive local folding transition upon DNA binding . Introduction of the mutation T93A at the DNA-binding cleft causes la rge-scale effects on the protein conformation. Structural changes in t he mutated unliganded enzyme propagate some 20 to 25 Angstrom to the d imerization interface and lead to a rearrangement of monomer subunits. Comparative analysis of these structures, a new structure of the enzy me cocrystallized with DNA and calcium ions, and previously determined cocrystal structures suggests important roles for a number of amino a cid residues in facilitating the intersubunit motions and local foldin g transitions. In particular, the T93A structure reveals a pathway thr ough 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 a re coupled to the protein conformational change. It appears that indir ect perturbation of the Glu45 conformation via an altered quaternary s tructure may be a contributing factor to the deceased catalytic effici ency of T93A, and this mechanism may also explain the diminished activ ities of other active site variants of EcoRV. (C) 1997 Academic Press Limited.