On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as thetarget

The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we em ployed a structure-guided approach to engineer variants of EcoRV that shoul d be able to discriminate between differently flanked EcoRV recognition sit es. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(A AAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flank ing the GATATC recognition site and thus were proposed to be a reasonable s tarting point for the rational extension of site specificity in EcoRV [Hort on,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729], To test t his proposal, several single (K104R, A181E, A181K) and double mutants of Ec oRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these vari ants prefer cleavage of a TA flanked site over all other sites, under all c onditions tested. The substitution of Lys104 by Arg, in contrast, which app eared to be very promising on the basis of the crystallographic analysis, d oes not lead to variants which differ very much from the EcoRV wildtype enz yme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E an d A181K single mutants. We conclude that even for the very well characteriz ed restriction enzyme EcoRV, properties that determine specificity and sele ctivity are difficult to model on the basis of the available structural inf ormation.