MvaI is a monomer according to analytical ultracentrifugation
At the sequence level, MvaI shows weak similarities to the monomeric DNA repair protein MutH (23
). This result prompted us to check the oligomerization state of MvaI in solution. Analytical ultracentrifugation shows MvaI to be mostly monomeric. Sedimentation velocity runs in the analytical ultracentrifuge with MvaI gave a sedimentation constant of s20°C,W
S. Using a mass for the monomeric protein of 28.6
kg/mol this corresponds to a frictional ratio of 1.27. For spherical hydrated proteins, a frictional ratio of 1.1–1.2 is expected (24
) and thus MvaI can be viewed as a mostly globular, monomeric particle. Sedimentation equilibrium gave a molar mass of 35
kg/mol indicating the protein to show some aggregation. This aggregation could be suppressed by the addition of 0.8
M GuaHCl where a molar mass of 27.5
kg/mol is observed.
MvaI is a monomer according to analytical gel filtration
The monomeric state of MvaI in the apo-form and in complex with the cognate, blunt-ended 9-bp oligoduplex 1 (A) was independently analyzed by analytical gel filtration. Experiments were run in the presence of calcium ions, which support DNA binding, but not hydrolysis (data not shown). Although MvaI and MvaI–DNA complexes are not resolved on the column, the amounts of MvaI and DNA in the elution peaks could be separately quantified at 260 and 280
nm, respectively, and deconvoluted based on the known A
(280) absorbance ratios for MvaI and DNA (see Materials and methods). For consistency, the deconvolution procedure was applied to all profiles, even if only protein or only DNA was injected (B–F).
In the experimental conditions (see Materials and methods), MvaI alone elutes from the column at 13.82
ml, which translates into a molecular mass of 30 ± 3
kDa, in agreement with the calculated monomer mass 28.6
kDa (B,G). The cognate 9-bp oligoduplex 1 (A) alone elutes much later, at 15.47
ml (C), but it coelutes with MvaI up to a stoichiometric ratio of 1 oligoduplex per 1 MvaI monomer (D and E). If oligoduplex is present in excess over the MvaI monomer, two peaks result. One equivalent of DNA coelutes with the protein, and the rest elutes as free DNA (F). The retention volume for the MvaI–DNA complex is 14.01
ml and corresponds to an apparent molecular mass value of 26 ± 2
kDa (G) which is slightly lower than the apparent mass of MvaI alone, probably because the more compact shape of the complex nearly balances the increase in molecular mass due to DNA binding. We conclude that MvaI remains monomeric in the presence of cognate DNA, at least in our experimental conditions. Our findings are inconsistent with a prior report of MvaI dimerization in the presence of both cognate and non-cognate DNA (25
MvaI binds duplex DNA in 1:1 stoichiometric ratio
The gel filtration experiments suggest that MvaI is a monomer that binds target duplex DNA in 1:1 stoichiometric ratio. This was independently confirmed by gel electrophoresis under acidic conditions (see, Materials and methods). In these conditions, free DNA does not enter the gel, but MvaI alone (predicted isoelectric point 6.8) migrates into the gel (Supplementary Figure 1, lane 1). The addition of substoichiometric amounts of cognate oligoduplex leads to the appearance of a second, slower migrating band, the MvaI–DNA complex (Supplementary Figure 1, lane 2). If oligoduplex is added in stoichiometric ratio, the band from MvaI alone almost disappears, and essentially only the MvaI–DNA band is present (Supplementary Figure 1, lane 3). Still further increase of the DNA concentration has no effect on the band pattern (Supplementary Figure 1, lane 4).
MvaI structure determination
The available biochemical data on MvaI and our findings about its oligomeric state and DNA-binding stoichiometry suggested that MvaI is a highly unusual restriction enzyme and prompted us to determine its structure. MvaI was crystallized in the apo-form and in the presence of oligoduplex 2 (). The crystals with and without DNA happened to be monoclinic and diffract to 1.5
Å resolution. The apo-form was solved by the SAD method, and the form with DNA by molecular replacement, using the previously determined model of the DNA-free form (details in Materials and methods and ).
Both crystal forms of MvaI contained two monomers in the asymmetric unit. In the apo-structure, the interaction of the monomers, which are related by a curious 2-fold symmetry, buries ~1800
of solvent-exposed surface. Despite this extensive contact, the interaction cannot be physiological, because (a) it is not observed in solution, (b) the interface is not conserved and (c) it locks MvaI in an open conformation that is not compatible with DNA binding (see below). In the crystals of the MvaI–DNA complex, the largest interfaces between adjacent molecules in the crystal bury only ~900 and ~600
. Moreover, these contacts relate molecules that cannot be mapped on each other by a simple 2-fold rotation, and they differ from the extensive contact in the apo-MvaI structure. Therefore, we conclude that MvaI crystallized as a monomer in all cases. The two molecules in the apo-MvaI structure are very similar, probably because the local 2-fold axis enforces it. Likewise, the two MvaI molecules in the asymmetric unit of the crystals with DNA overlap almost perfectly, probably because the complexes with DNA are very compact and therefore rigid. The same is true for the bound DNA duplexes, which can be described by very similar conformational parameters according to the 3DNA program (19
) (Supplementary Table 1), even though no restraints or constraints were applied during refinement. For simplicity, we will not distinguish between the two monomers in each crystal form in the following text.
MvaI consists of two lobes
MvaI is organized into two lobes which we term the catalytic lobe (residues 1–63, 160–188 and 238–246, orange in ) and the recognition lobe (residues 64–159 and residues 189–237, green in ) based on their mechanistic roles discussed below. The orientation of the two lobes differs radically depending on whether or not DNA is bound: in the absence of DNA, the molecule has an almost flat appearance (A), but in the presence of DNA, it forms a tight ‘clamp’ around it (B). Despite the drastic change in hinge angle (~54°), there is no major conformational difference within the lobes, except in loops (C and D and Supplementary Figure 2). The rearrangement of residues 40–57 is significant, because this region includes catalytic residues.
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 (more ...)
The catalytic lobe is organized around a four-stranded mixed β-sheet which is flanked by two α-helices and a 310
-helix (orange part of ). The topology of the sheet is +1x, +1, +1, +1 according to the Richardson nomenclature (26
). Note that elements of the recognition lobe are interspersed between strands βC1 and βC2 and also between strands βC3 and βC4. Strands βC2 and βC3 are connected by a simple hairpin. The fold of the catalytic lobe is fairly similar to the fold of its counterpart in MutH, which has been termed the ‘N-arm’ of this enzyme (27
). There are also other, more distant similarities to the cores of other REases of the PD … EXK family (data not shown).
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. (more ...)
The term ‘catalytic’ lobe was chosen because this part of MvaI anchors all catalytic residues of the enzyme. In addition, it also contributes some of the minor grove interactions with DNA, which are unlikely to play a major role in sequence discrimination (see below).
In contrast to the catalytic lobe, the recognition lobe is characteristic of MvaI. The fold is organized around two antiparallel β-sheets (green part of ). The larger of the two sheets consists of strands βR1, βR2, βR3, βR7 and βR8/βR9, which are connected in +4x, −1, −1, −1 topology. This would correspond to the Greek key motif if the small βR2 is not taken into account. The smaller sheet is built from strands βR4, βR5 and βR6 and is a β-meander. Protein architecture dictates two preferred ways for β-sheets to stack against each other: aligned, with an angle between the strands in the two sheets ~30°, and perpendicular, with an angle between the strands ~90° (28
). The MvaI recognition lobe clearly belongs to the latter group and therefore resembles a half-barrel or barrel (30
) except for the lack of hydrogen bonds to connect the sheets.
The term ‘recognition’ lobe was chosen because this lobe anchors the residues that interact specifically with the major grove of DNA and likely mediate sequence discrimination. We also note that nearly all basic residues (Lys64, Lys72, Arg85, Lys90, His100, Arg107, Lys159, Lys205, His214) that interact with the phosphodiester backbone of DNA are located in the recognition lobe.
One active site, asymmetric recognition of the pseudosymmetric target sequence
MvaI acts as a monomer and recognizes its pseudosymmetric target sequence asymmetrically ( and B). As the enzyme has only one active site, this implies that only one strand can come into proximity of the active site. Although MvaI can bind target DNA in two orientations, the strand with the central T (‘T-strand’) binds exclusively or predominantly close to the active site in our crystal form of MvaI with DNA.
As expected, MvaI contacts the specifically recognized target bases, but in addition it also engages in hydrogen bonds with a flanking base pair. In all positions, the catalytic lobe approaches the bases exclusively from the minor grove side, and the recognition lobe interacts with the bases exclusively from the major grove side. For the detailed discussion, we follow the T-strand from the −3 to the +2 position (from left to right according to the scheme in ).
A − 3 T + 3
: This A-T base pair is not part of the recognition sequence, but it nevertheless makes two direct hydrogen bonds with MvaI. The O2 and O4 atoms of thymine accept hydrogen bonds from the side chain amide group of Asn45 and the OH of Tyr213, respectively. From the structural perspective, it can be expected that these interactions contribute little to sequence specificity: the position of the O2 atom, the so-called ‘outer minor grove’ is taken by a hydrogen bond acceptor for all four possible base pairs (32
). On the major grove side, the side chain oxygen atom of Tyr213 can act as a hydrogen bond donor as in the crystallographically observed complex, but might also act as a hydrogen bond acceptor, if other base pairs are present in this position (A).
Figure 5. Hydrogen-bonding interactions between MvaI and DNA. Panels are ordered following the T-strand in 5′ → 3′ direction as indicated in . Residues of the catalytic lobe are labeled in orange, and residues of the recognition (more ...) C − 2 G + 2
: This C-G base pair forms only indirect hydrogen bonds with MvaI on the minor grove side, but is involved in two direct hydrogen-bonding interactions with the enzyme on the major grove side. The N4 atom of cytosine donates a hydrogen bond to the main chain carbonyl oxygen atom of Asp224 and the O6 atom of guanine accepts a hydrogen bond from the Nε atom of His223. This interpretation requires that His223 is either charged or is in the tautomeric state with the proton on the N
atom (B and data not shown).
C − 1 G + 1: This C-G base pair makes an indirect hydrogen bond to Arg25 on the minor grove side and interacts with His225 on the major grove side. The main chain carbonyl oxygen atom of this residue accepts a hydrogen bond from the N4 atom of cytosine, and the Nδ atom of its imidazole ring donates a hydrogen bond to the O6 atom of guanine. For the interaction between the imidazole ring and the base to be sequence selective, the tautomerization state of His225 should be locked by interactions within MvaI, and not just by the hydrogen bond to the base. As the Nε atom of His225 interacts with a water molecule that could be either a donor or an acceptor, if and how this ‘lock’ is provided remains unclear (C and data not shown).
T0 A0: This T-A base pair accepts a direct hydrogen bond from Thr29 Oγ to the thymine O2 atom and an indirect hydrogen bond to the adenine N3 atom. As the ‘outer minor grove’ positions are taken by hydrogen bond acceptors for all possible base pairs, this interaction probably contributes little to specificity. On the major grove side, Arg209 donates a hydrogen bond to the O4 oxygen atom of the T-strand thymine in the crystallized MvaI–DNA complex. There is no trace of the alternative binding mode, which swaps purine and pyrimidine and must occur as well in solution (D).
G + 1 C − 1: This G-C base pair is involved in extensive hydrogen-bonding interactions. On the minor grove side, Asn28 accepts a direct hydrogen bond from the N2 atom of guanine and anchors a water molecule that donates a hydrogen bond to the O2 atom of cytosine. On the major grove side, the carboxylate of Asp207 accepts a direct and a water-mediated hydrogen bond from the cytosine N4 atom. The guanidino group of Arg209 and Thr68 Oγ atoms donate hydrogen bonds to the O6 and N7 atoms of guanine, respectively (E).
G + 2 C − 2: This G-C base pair makes only major grove interactions with MvaI. The guanidino group of Arg230 donates hydrogen bonds to the guanine atoms O6 and N7, and the Thr102 Oγ atom accepts a hydrogen bond from the cytosine N4 atom (F).
The methylation sensitivity of MvaI has been extensively studied with special emphasis on the differential effects of methylation on the hydrolysis of the two DNA strands. Experimentally, it was found that 5mC can replace cytosine in all positions (Supplementary Table 2), but this would have been difficult to predict from the crystallographic results. In silico
introduction of 5mC instead of the outer and inner cytosines of the T-strand brings the extra methyl groups within 2.9
Å of Gly226
Cα and within 3.0
Å of His225 O, respectively, if base positions are not adjusted and the enzyme is kept rigid. Similarly, in silico
conversion of the cytosines to 5mC in the A-strand would introduce methyl groups 2.3
Å away from Thr102 Oγ and 3.3
Å away from His100
Cβ, respectively, and would additionally require the displacement of a water molecule. Therefore, the experimentally observed tolerance of MvaI to 5mC has to be attributed either to flexibility of the enzyme, or alternatively to the adjustability of the exact positions of the bases (data not shown).
In contrast to the effects of C5 methylation, the consequences of N4 methylation on T-strand cleavage can be readily explained by the crystal structure. N4mC cannot replace the inner cytosine in either strand, because the extra methyl group would clash with a hydrogen bond acceptor of the protein (His225 O, C and Asp207 Oδ, E). Likewise, N4mC in the outer position of the T-strand is not tolerated, again because the methyl group would clash with a hydrogen bond acceptor on the protein (Asp224 O in this case, B). Experimentally, substitution of the A-strand outer cytosine with N4mC has been reported not to interfere with cleavage: apparently the methyl group can displace the side chain of Thr102 (F).
Substitution of the central adenine with N6mA interferes with A-strand cleavage, but does not affect T-strand cleavage. The latter result is consistent with the crystal structure, because the methyl group of N6mA only needs to displace a water molecule to fit in (D).
The rules for T-strand cleavage have direct implications for A-strand cleavage, and therefore the above results can be summarized in four simple rules: (a) substitution of cytosine with 5mC has no effect. (b) Substitution of the inner cytosine with N4mC in one strand blocks cleavage of both strands. (c) Substitution of an outer cytosine in one strand by N4mC abolishes cleavage of this strand, but does not interfere with cleavage of the complementary strand. (d) Substitution of the central adenine with N6mA affects A-strand, but not T-strand cleavage. Together, these four rules correctly predict the outcome of a large number of experiments on the methylation sensitivity of MvaI (Supplementary Table 2).
Note that the MvaI MTase methylates the N4 atoms of the inner cytosines. Rule (b) predicts correctly that this modification interferes with DNA cleavage. Conversely, the Dcm MTase converts the same cytosine to 5mC, which should not have an effect on DNA cleavage. This is consistent with the experimental observation that DNA from dcm+ strains can be cleaved by MvaI (Supplementary Table 2).
MvaI active site
MvaI crystals with and without DNA were grown in the absence of Mg2+, but in the presence of Ca2+ ions, which support DNA binding (see ), but not DNA hydrolysis (data not shown). In the DNA-free form, Cd2+ ions were present in addition to the Ca2+ ions, but no metal ions were found in the vicinity of the active site residues. This unexpected result is due to the arrangement of residues 40–57, which are present in radically different conformations in the apo-MvaI structure (A) and in the complex with DNA (B).
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 (more ...)
In the productive orientation two metal-binding sites are formed, which are occupied by Ca2+ ions from the buffer (the identification of the metals is supported by the ligand distances and the X-ray anomalous signal). In both standard electron density maps and anomalous difference Fourier maps, the peak heights for the two metals are very different. The weaker peak (yellow ball in C) corresponds to a Ca2+ liganded to an oxygen atom of the scissile phosphate, one Asp50 Oδ atom and three or four water molecules (depending on which molecule in the asymmetric unit is used for the analysis). The stronger peak corresponds to a hexa-coordinated Ca2+ ion with an almost perfect octahedral coordination sphere. The ligands to this metal ion are the other Oδ oxygen atom of Asp50, the Oε atom of Glu55, the main chain carbonyl oxygen atom of Ile56, an oxygen atom of the scissile phosphate, which acts as a bridge between the two metals, and two solvent molecules (C).
One of these solvent molecules, which could either be a water molecule or a hydroxide ion (shown with its electron density in C) is positioned exactly on the line that links the O3′ oxygen atom of the C−1 residue to the phosphorus atom of the scissile phosphate. This solvent molecule is ideally positioned for an in-line attack on the phosphate atom, which might proceed via a bipyramidal transition state and would correctly predict reaction products with a free 3′-end and a phosphorylated 5′-end.
Although the above catalytic mechanism appears plausible, the reaction does not proceed in the crystals, as evidenced by several detailed features of the crystal structure. (a) There is robust electron density for the potentially scissile phosphorus oxygen bond, suggesting that this bond is predominantly not cleaved. (b) At least at the present resolution, there is no significant deformation of the tetrahedral geometry at the phosphorus atom towards a trigonal bipyramidal arrangement. (c) The putative ‘catalytic’ water molecule is 3.3
Å away from the phosphorus atom. This distance is not significantly smaller than the sum of the van der Waals radii, if coordinate errors of the crystal structure are taken into account, but it is significantly larger than a typical phosphorus oxygen bond in DNA (~1.6
Å). Therefore, it seems that the reaction has either not started in the crystal structure, or alternatively that it is trapped very early in the trajectory, which is consistent with the biochemical observation that Ca2+
ions in the active site support DNA binding, but not DNA cleavage. Why is the reaction blocked with Ca2+
ions in the active site and would proceed with Mg2+
ions in the active site? Typical oxygen ligand distances are much shorter for Mg2+
than for Ca2+
Å versus 2.4
), which might lead to a slight mispositioning of the nucleophilic water molecule in the complex with Ca2+
. However, more sophisticated simulation studies attribute the different efficiencies of the Mg2+
forms of REases to kinetic effects and not to the properties of the prereactive states of these enzymes (34