A new crystal form of restriction endonuclease EcoRII that diffracts to 2.8 Å resolution

Abstract

EcoRII, a type IIe restriction endonuclease, has been crystallized in space group P2 1 , with unit-cell parameters a = 58.3, b = 127.8, c = 59.9 A Ê , = 91.4. There are two monomers in the asymmetric unit and the solvent content is estimated to be 49% by volume. The crystals diffract to 2.8 A Ê resolution, which is much higher than that of the previously reported cubic crystal form, which diffracted to 4 A Ê resolution.

Crystal Structure of Type IIE Restriction Endonuclease EcoRII Reveals an Autoinhibition Mechanism by a Novel Effector-binding Fold

“…32 In contrast, a single amino acid substitution at position 88 from arginine to alanine (R88A) led to better crystals that diffracted to 2.1 Å resolution and were easier to grow. 33 Screening a membrane-bound Eco RII-derived peptide scan with an Eco RII recognition site containing oligonucleotide duplex we had identified two separate, potential DNA-binding regions of Eco RII, amino acid residues 88 -102 and 256 -273, which share the concensus motif KXRXXK and show an abundance of the positively charged amino acids, Arg and Lys. 27 Arg88 is located at the beginning of DNA-binding region I.…”

“…33 In brief, expression and purification procedures for R88A were the same as those used in the wild-type production. 27,32 Crystals were grown at room temperature by the hanging-drop vapor-diffusion method with 100 mM cacodylate buffer (pH 6.3 -6.5), 3.5-4% (v/v) methanol, 35 -40 mM MgCl 2 as crystallization solution.…”

Type II restriction endonucleases: structure and mechanism

“…Type IIE enzymes need to interact with two copies of their recognition sequence for efficient cleavage, one copy being the target for cleavage, the other serving as an allosteric effector [28][29][30]. The best-studied examples with respect to structure and function are EcoRII (/CCWGG) [31][32][33][34][35][36] and NaeI (GCC/CGG) [37][38][39][40][41]. It is interesting to note that the removal of the effector domain of EcoRII converts this Type IIE enzyme into a very active Type IIP enzyme [34].…”

Structure and Function of Type IIE Restriction Endonucleases — or: From a Plasmid That Restricts Phage Replication to A New Molecular DNA Recognition Mechanism

“…In the absence of Mg2+, cubic EcoRII crystals were obtained which diffracted to about 4.0 A (Karp ova et al 1999b). In another attempt using His.-tagged EcoRII, monoclinic crystals appeared in the presence of Mg2+ and diffracted to 2.8 A (Zhou et al 2002). Both EcoRII wild-type crystals revealed two EcoRII monomers per asymmetric crystal unit.…”

“…The crystals were easier to grow and diffracted to 2.1 A. Their structure was now determined (Zhou et al 2003(Zhou et al , 2004). The overall EcoRII structure consists of 18 a-helices and 13 p-strands and can be divided into two domains linked through a hinge loop.…”