Restriction endonuclease MvaI is a monomer that recognizes its target sequence asymmetrically

Abstract

Restriction endonuclease MvaI recognizes the sequence CC/WGG (W stands for A or T, '/' designates the cleavage site) and generates products with single nucleotide 5'-overhangs. The enzyme has been noted for its tolerance towards DNA modifications. Here, we report a biochemical characterization and crystal structures of MvaI in an apo-form and in a complex with target DNA at 1.5 A resolution. Our results show that MvaI is a monomer and recognizes its pseudosymmetric target sequence asymmetrically. The enzyme consists of two lobes. The catalytic lobe anchors the active site residues Glu36, Asp50, Glu55 and Lys57 and contacts the bases from the minor grove side. The recognition lobe mediates all major grove interactions with the bases. The enzyme in the crystal is bound to the strand with T at the center of the recognition sequence. The crystal structure with calcium ions and DNA mimics the prereactive state. MvaI shows structural similarities to BcnI, which cleaves the related sequence CC/SGG and to MutH enzyme, which is a component of the DNA repair machinery, and nicks one DNA strand instead of making a double-strand break.

Figure 1.

Analytical gel filtration experiments to determine the MvaI oligomeric state and the stoichiometry of DNA binding for the oligoduplex 1 shown in (A). Elution profiles were recorded simultaneously at 260 and 280 nm and deconvoluted to obtain separate curves for the MvaI (blue) and DNA (red) concentration. (B) MvaI alone, (C) DNA alone, (D) mixture with a 2:1 molar excess of MvaI over DNA, (E) stoichiometric mixture, (F) mixture with a 2:1 molar excess of DNA over MvaI, (G) calibration curve for Superose ™ 12 HR 10/30 column (Amersham Biosciences) with standards from Biorad (vitamin B-12, 1.35 kDa; myoglobin, 17 kDa; ovalbumin, 44kDa; IgG, 150 kDa and thyroglobin, 670 kDa).

Figure 2.

Summary of MvaI–oligoduplex 2 interactions. Residues of the catalytic and recognition lobes are marked in orange and green, respectively. Arginine, lysine and histidine residues of MvaI involved in the interactions with DNA phosphate oxygen atoms are indicated close to the respective phosphates. Direct hydrogen bonding interactions of MvaI with the DNA bases are indicated next to the bases, on their right side (e.g. Y213 and N45 interact with T + 3). Indirect water-mediated hydrogen bonding interactions are not shown. The black arrows indicate the MvaI cleavage sites. The binding mode of the DNA in the MvaI crystals brings only the T-strand close to the active site. Labels A, B, C, D, E and F refer to the panels in Figure 5.

Figure 3.

Overall view of the MvaI structure. The catalytic lobe of MvaI is shown in orange and the recognition lobe is presented in green. (A) Ribbon representation of the open conformation in the apo-form of MvaI. Rotation of the recognition lobe around the blue axis by 54° brings this lobe into a similar orientation relative to the catalytic lobe as in the MvaI–DNA complex. (B) Ribbon representation of the closed conformation in the MvaI–DNA complex. The DNA strand that comes close to the active site and would be cleaved in the presence of Mg²⁺ ions is shown in dark red. The complementary strand is presented in black. (C) Superposition of the Cα-traces of the catalytic lobes taken from the structures without (orange) and with (gray) DNA. (D) Superposition of the Cα-traces of the recognition lobes from the structures without (green) and with (gray) DNA. All panels were prepared with the MOLSCRIPT program (44).

Figure 4.

Schematic representation of the MvaI fold. The catalytic domain is in orange and the recognition domain is in green. Catalytic residues are marked by black dots, and residues that are involved in hydrogen-bonding interactions are marked by colored circles. Colors are ramped from red to blue following the T-strand in 5′ → 3′ direction. Filled circles mark residues that interact with the T-strand and open circles indicate residues that interact with the A-strand in the crystal structure. The secondary structure assignment was done for the structure with DNA using the DSSP program (45): αC1 (L5-V16), αC2 (G30-L38), βC1 (I54-E60), 3₁₀C (T61-A63), βR1 (L67-T71), αR1 (A80-Y88), βR2 (K90-K91), βR3 (N97-V103), βR4 (Y116-D123), βR5 (V128-D135), βR6 (M140-S148), αR2 (F149-K159), βC2 (Y162-E173), βC3 (K176-T188), αR3 (V192-N201), βR7 (I204-A212), βR8 (T222-D224), βR9 (A228-I231), 3₁₀R (M233-E238) and βC4 (E241-V244).

Figure 5.

Hydrogen-bonding interactions between MvaI and DNA. Panels are ordered following the T-strand in 5′ → 3′ direction as indicated in Figure 2. Residues of the catalytic lobe are labeled in orange, and residues of the recognition lobe are in green. Hydrogen bonds are indicated by dotted lines. Prior experimental results on the effects of single-strand methylations are indicated below the interaction diagrams. A ‘+’ sign indicates that the methylation is compatible with T-strand cleavage, and a ‘−’ indicates that the corresponding methylation reduces T-strand cleavage by at least 50%. The abbreviations (a), (b), (c), (d) refer to the following publications. (a) Gromova et al. (1991) (10) (b) Butkus et al. (1985) (6) (c) Kubareva et al. (1988) (7) (d) Kubareva et al. (1990) (46).

Figure 6.

MvaI active site: (A) Conformation in the crystal of the apo-form. (B) Conformation in the cocrystals with DNA and Ca²⁺ ions. In (B), the yellow and orange balls represent the two Ca²⁺ ions in the structure, and the dark red curve is the T-strand of DNA in ribbon representation. (C) Stereo representation of the active site of MvaI in the form with bound DNA. The 2fofc density has been contoured at 1.2σ and is shown around the DNA and the catalytic water molecule or hydroxide ion. A productive nucleophilic attack, which does not take place in the crystals would require an approach of the catalytic water molecule or hydroxide ion towards the phosphorus atom of the scissile phosphate along the green line. Glu36 at the back of the figure has not been labeled to avoid overlap.