We crystallized a ternary complex containing HinP1I, a 10 bp oligonucleotide containing a single GCGC site, and a divalent metal ion, Ca2+
, in the P32
21 space group (PDB ID 2FKC). The crystallographic asymmetric unit contains two HinP1I molecules (A and B), forming a back-to-back dimer as observed in the structure of free HinP1I (5
), and two DNA duplexes ()—strands C and D are bound to molecule A and strands E and F are bound to molecule B.
Figure 1 HinP1I–DNA–Ca2+ structure. (A) The back-to-back HinP1I dimer bound with two DNA duplexes. The HinP1I monomer is colored using a rainbow spectrum with blue for the N-terminus and red for the C-terminus. (B) A HinP1I monomer bound with one (more ...)
The 10 bp DNA duplex is encircled by a single HinP1I molecule (); with the long 7-turn N-terminal helix in the minor groove and the β-strands and their associated loops in the major groove. Thus, ~1900 Å2
of surface area per protein is buried upon DNA binding, comparable with what is observed for the monomeric mismatch repair endonuclease MutH when it binds DNA (9
). Also, the bound DNA is substantially bent, ~60° (), by its interaction with the enzyme (see below). The bent duplex stacks with one neighboring DNA molecule at each end forming a superhelix. The axis of the superhelix formed by the assembly of the oligonucleotide is parallel to the crystal c
-axis and the DNA obeys the 32
screw-symmetry of the space group with a radius of ~50 Å (half of the unit cell a
-axis) and a rise of 130 Å (the length of the unit cell c
-axis), giving a repeat of 60 bp. The assembled superhelices are connected via two HinP1I molecules through the back-to-back dimer interface (), defining the packing of the protein–DNA complexes that complete the formation of the crystal. The HinP1I–DNA packing is very similar to the DNA condensation induced by some DNA bridge factors, which use two DNA binding motifs located on opposite surfaces of a bridge dimer (10
HinP1I changes conformation upon DNA binding
DNA molecules bound by face-to-face dimeric REases are almost all accommodated in a tight binding cleft formed by both monomers (2
). In the cases of HinP1I and MspI (3
), the DNA binding cleft lies within a single protein molecule, which raises the question whether a conformational change in the protein is necessary for tight binding. Comparing the structures of free and bound HinP1I, the largest change involves in the N-terminal 17 residues, which become part of a long helix upon binding to cognate DNA (). In the structure of free HinP1I, the first six N-terminal residues that are invisible in the electron density map are followed by a β-strand (residues 9–11) and a loop (residues 12–17), which is connected to the helix αA (5
). Upon binding to DNA, helix αA is extended to the very first residue and the resulting longer helix clamps down along the minor groove of the DNA (). This results in a local A-form geometry around the two central C:G base pairs of the recognition site: a wider minor groove (~10 Å width versus 6 Å in B-form), a shorter base rise distance (~2.4–2.9 Å versus 3.4 Å in B-form) and a tilting of the bases (~10–20°) rather than lying perpendicular to the helix axis (). Interestingly, the residue adjacent to this helix extension, E18, forms part of the catalytic center (see below). Comparing the two structures, with and without DNA bound, there is very little change in the catalytic center, suggesting that it is pre-formed and relatively rigid.
Local inter-base parameters of DNA
DNA distortion by side chain intercalation and base flipping
The major disruption to the base stacking and linearity of the DNA occurs at the two junctions of the recognition site. On one distal side of the recognition site, between the outer base pair of the recognition site (C:G at position 7) and the first base pair of the flanking DNA (T:A at position 8) (), the hydrophobic side chain of F91 intercalates the DNA from the major groove causing the DNA to be kinked by ~60° (). The intercalation expands the base rise distance to ~4.7 Å between the base pairs at positions 7 and 8 (). On the proximal side of the recognition site, the Gua of the outer G:C base pair (at position 4) is in van der Waals contact with the phenyl ring of F15 of the N-terminal helix αA, which approaches the DNA from the minor groove (). This asymmetric intercalation is similar to the intercalation of the repair proteins MutS and Vsr (which function as monomers) with DNA mismatches (12
), but differs from the symmetric intercalation of the HincII restriction endonuclease, a face-to-face dimer, in which a Gln side chain intercalates between 2 bp on either side of the recognition site (16
Figure 2 HinP1I–DNA interactions. (A) Summary of the protein–DNA contacts of HinP1I (green). Backbone mediated interactions are indicated with main chain amine (N) or carbonyl (O). For simplicity, only single water (w) molecule mediated interactions (more ...)
The most striking finding in the structure is that Ade3, immediately outside of the proximal side, can adopt at least two conformations, extrahelical and intrahelical. Shown in is a least-squares superimposition of two DNA duplexes (strands C and D versus strands E and F), using the protein component only to determine the superimposition (a root-mean-square deviation of ~0.6 Å comparing 247 pairs of Cα atoms of two HinP1I molecules A and B). The two duplexes show high concordance in the interaction pattern of the recognition base pairs (at positions 4–7), and the distal T:A base pair (at position 8) immediately adjacent to the F91 intercalation site. On the other side of the recognition sequence, the helix conformation of the first flanking base pair, A:T (at position 3 of ), is markedly different in the two duplexes. In molecule A, the A:T base pair at position 3 remains stacked and hydrogen bonded in the duplex (left boxed enlargement in ). In molecule B, the Ade of the A:T base at position 3 is flipped out of the DNA helix (right boxed enlargement), where it is stabilized extrahelically by residues H97, W98, M234, and the 5′ Cyt base at position 2 (). Interestingly, the local DNA structure adopts features characteristic of Z-form DNA between the base pairs at positions 3 and 4 (), reminiscent of the recent finding of base flipping at the junction between B- and Z-DNA (17
). An important parameter is a relatively long rise distance of 5.7 Å between the T(-3) and C(-4) bases (strand F in ), which may be attributable to the flipping of the A(3) base of strand E, which has a twist angle of −13°, an indication of a transition from B-form (36°) to Z-form (−30°). The corresponding rise distance between the base pairs 3 and 4 in strand C and D is slightly smaller, one possible reason why A(3) base of strand C stays stacked. Compared with the two extruded bases at the junction between B-DNA and Z-DNA () (17
), the A(3) base lies in a position similar to the flipped thymine at the B-to-Z junction. Both flipped bases, A(3) in HinP1I and the thymine at the B-to-Z junction, have a stabilizing force via a face-to-edge van der Waals contact with an intrahelical base (comparing ), while the fully extended adenine at the B-to-Z junction has neither contacts with the protein nor the DNA (17
Figure 3 Base flipping outside of the recognition sequence. (A) Superimposition of the two DNA duplexes, bound with molecule A (colored in grey) or molecule B (colored with yellow for carbon atoms, blue for nitrogen atoms, red for oxygen atoms and magenta for (more ...)
We also obtained a second crystal form of HinP1I–DNA–Ca2+ (see Materials and Methods) where the A(3) base in every duplex in the crystal is flipped out (data not shown). The new crystal form can be indexed in a higher symmetry space group P6522 where the crystallographic asymmetric unit is reduced to containing one Hin1PI molecule and one DNA duplex (PDB ID 2FKH). The protein component of pre-reactive complex in the space group P6522 is more similar in structure to the extrahelical-A(3)-containing molecule B (root-mean-square deviation of ~0.3 Å) than the intrahelical-A(3)-containing molecule A (root-mean-square deviation of ~0.6 Å) in the space group P3221. The extrahelical-A(3) in both space groups, P3221 and P6522, is not involved in any crystal packing contacts. We suggest that the degree of deviation of local structure around A(3), away from B-DNA toward Z-DNA, determines whether A(3) flips.
Enzyme-induced DNA base flipping has been characterized structurally in DNA methyltransferases and glycosylases (18
) and in a DNA polymerase (21
). In these cases, the flipped nucleotide is either the target of modification or repair or it is the template nucleotide for DNA synthesis. In solution, incorporation of the nucleotide analog 2-aminopurine (2AP) into synthetic oligodeoxynucleotide duplexes has been used to probe conformational changes, such as base flipping (22
), because 2AP fluorescence increases dramatically when it is removed from the stacking environment of double helical DNA (27
). However, in some cases, the 2AP fluorescence change does not correlate with the target of modification or repair (28
), when the 2AP was positioned at a non-target site or outside of recognition sequence. The ambiguity calls into questions whether 2AP fluorescence is a reliable diagnostic technique for DNA base flipping in solution. While the technique is being fine tuned, our structure of a HinP1I–DNA complex provides the first example of a nucleotide outside of a recognition sequence that can be either intrahelical or extrahelical upon binding of the DNA to protein. It should be noted that when MspI binds to an oligonucleotide of the same length as used here, it retains the typical B-form DNA structure (3
). There are no equivalents in MspI to F15 and F91 in HinP1I.
Direct major groove protein–DNA contacts
In addition to phosphate interactions, which span 6 bp (), all eight bases of the tetranucleotide recognition sequence GCGC have direct hydrogen bond interaction with one HinP1I molecule. The interactions with the four guanines are all through their O6 atoms, while the four cytosines are contacted through their N4 atoms (). Three lysine side chains (K96, K223 and K238) and one glutamine side chain (Q236) are involved in the interactions with the guanines. Two side chain oxygen atoms (D226 and Q93) and two main chain carbonyl oxygen atoms (K223 and F91) are involved in the interactions with the cytosines.
Pre-reactive ternary complex of HinP1I–DNA–Ca2+
ions (m1 and m2, separated by 4.2 Å distance) were identified in the active site: both have octahedral coordination (). Metal m1 is coordinated by the side chain oxygen atoms of D62 (Oδ2
, 2.5 Å) and Q81 (Oε1
, 2.5 Å), the main chain carbonyl oxygen of V82 (2.4 Å), the oxygen O1P of the scissile phosphate (2.1 Å) and a water molecule w1 (2.4 Å). Besides making contact with the metal m1, the water molecule w1 is hydrogen bonded to the O1P oxygen of the scissile phosphate (3.0 Å) and the O2P oxygen of the 3′ nucleotide (2.6 Å). The water molecule w1 is well positioned, 3.4 Å from the phosphorous atom and makes an angle of ~167° with the P-O3′ bond of the scissile phosphate group, to act as the attacking nucleophile for an in-line attack opposite the O3′ leaving group. It is unclear which component of the catalytic site is responsible for activating the nucleophilic water molecule. The side chain amino group of K83, a highly conserved catalytic site residue among restriction enzymes but is replaced by Glu in BamHI or Gln in BglII, appears to be too far removed from the water (4.0 Å) to accept the proton. If the Lys has any role in activating the water molecule, as proposed for many restriction enzymes, deprotonation of K83 would be critical. Because the typical pKa
of a free Lys side chain is 10.8, the proper local environment (such as an immediately proximal positive charge or a hydrophobic microenvironment) might lead to a significantly depressed pKa
value for K83. In the bacteriophage T5 flap endonuclease, the positively charged metal-ion cofactor lowered the pKa
value of the ternary complex to 8.3 (with Mg2+
as a cofactor), 7.0 (Mn2+
) or 6.0 (Co2+
). As an alternative, the negatively charged pro-Rp oxygen 3′ to the scissile bond might play a substrate-assisted catalytic role by accepting the proton [as proposed for EcoRI and EcoRV (32
), MutH (9
) and RNase H (33
)], although its pKa
, conversely, would ordinarily be too low.
Figure 4 Two-metal mechanism. (A) In the pre-reactive complex, two Ca2+ ions (m1 and m2) are bound in the active site. The dashed lines indicate hydrogen bonds. (B) Structural superimposition of the active site of HinP1I (green) with that of MutH [cyan; PDB 2AOQ (more ...)
Metal m2 is bound by the side chain oxygen atoms of D62 (Oδ1
, 2.5 Å) and E18 (Oε1
, 2.6 Å), the leaving group O3′ oxygen of 5′ Gua (2.6 Å), the oxygen O1P of the scissile phosphate (2.9 Å) and a water molecule w2 (2.6 Å). The metal-associated water molecule w2 is 3.3 Å away from the 3′-oxygen leaving group and could function as a general acid by protonating the leaving group. The geometries of the two metal ions and their associated water molecules support the two-metal catalytic mechanism (2
A common catalytic site motif among restriction enzymes is characterized as PDXn(D/E)XK, with the consensus residues clustering around the scissile phosphate (2
). The corresponding catalytic motif in HinP1I and MspI is (S/T)DX17-18
). The active site of HinP1I is most similar to that of MutH (), all elements involved in the active-site formation are superimposable including the two Ca2+
metal ions, the attacking water molecule, the ion coordination, the scissile phosphate and the 3′ pro-Rp oxygen. One major difference is the replacement of E77 of MutH (EXK) by Q81 of HinP1I (QXK). The Nε2
atom of side chain of Q81 hydrogen bonds to the main chain carbonyl oxygen of K60 (), an interaction that might confer additional stability to the active site. A similar interaction is observed between the side chain of N117 of MspI (NXK) and the main chain carbonyl of K97. However, no corresponding side chain–main chain interaction exists in MutH or in the active sites of REases involving (E/D)XK.
DNA cleavage in solution
The back-to-back dimer of HinP1I bound to two DNA duplexes (), with only one active site for each duplex (), raises the interesting question of whether HinP1I cleaves the two strands of duplex DNA separately. To answer this question, we tried to cleave DNA both in the crystal (see below) and in solution to see whether HinP1I generates a nicked intermediate. We used supercoiled pUC19 plasmid DNA (2.7 kb, with 17 HinP1I sites) as template. shows a time course of digestion with a 1:4 molar ratio of HinP1I to pUC19 DNA. The earliest product was a nicked open circle intermediate, which accumulated before being converted to a linear product, consistent with HinP1I cleaving DNA one strand at a time. In comparison, very little nicked open circle was produced by EcoRI (), while more nicked intermediate was produced by BamHI, an enzyme known to act on the two strands asymmetrically (35
). Although HinP1I clearly formed a nicked intermediate, the amount of it seemed to be less, and the appearance of the linear product was earlier, than one would expect from a completely random nicking reaction, considering that pUC19 contains 17 HinP1I sites. This might suggest that the likelihood of strand-hydrolysis at any HinP1I site increases greatly once the other strand is already hydrolyzed. Either HinP1I remains in the vicinity of its site after hydrolysis and can rebind it with high probability, or hydrolysis of the second strand at any site proceeds more rapidly than does hydrolysis of the first.
Figure 5 Digestion of supercoiled (SC) DNA by HinP1I in solution. (A) Each 5 µl reaction in the New England Biolabs (NEB) buffer 3 (50 mM Tris, pH 7.9, 10 mM MgCl2, 100 mM NaCl and 1 mM DTT) contains 0.16 µg of pUC19 DNA and 1 ng of HinP1I (a 1:4 (more ...)
Metal-free binary complex of HinP1I–DNA
To investigate DNA cleavage in the crystal, we first crystallized the HinP1I–DNA complex in the absence of metals, in the space group P6522 (PDB ID 2FL3). One HinP1I (molecule C, to distinguish it from the A and B molecules in the pre-reactive complex) and one DNA duplex were present in the asymmetric unit. However, the HinP1I monomer interacts with its neighboring protein molecule using the same back-to-back dimer interface via a crystallographic 2-fold symmetry. The binary structure is highly similar to that of the pre-reactive ternary complex. Shown in is a superimposition of the two active sites both bound with DNA, using the protein component only to determine the superimposition (root-mean-square deviation of ~0.35 Å comparing 247 pairs of Cα atoms between molecules A and C). A single water molecule was found in the active site,occupying a position ~1.5 Å away from the first metal site m1. The water molecule is coordinated via three strong hydrogen bond interactions (~2.7 Å) with the side chain oxygen of D62 (Oδ2), the main chain carbonyl oxygen of V82, the oxygen O1P of the scissile phosphate and a weak hydrogen bond interaction (3.3 Å) with the side chain oxygen of E18 (Oε1).
Post-reactive complex of HinP1I: two-metal catalytic mechanism
We soaked pre-formed crystals of the metal-free binary complex of HinP1I–DNA in mother liquors containing Mg2+ ions. Crystals usually cracked or disintegrated. After much trial and error, and careful control of metal concentration, soaking times and the size of crystals (see Materials and Methods), we were able to collect a dataset at 2.6 Å resolution () (PDB ID 2FLC).
Remarkably, the DNA is cleaved in the crystal showing that the back-to-back dimer maintains a catalytically active conformation in the crystalline state. DNA cleavage occurs only in one strand and the active site contains two Mg2+
ions (). The overall root-mean-square deviation between the Cα positions of the pre-reactive Ca2+
complex (molecule A) and the post-reactive Mg2+
complex is only ~0.5 Å. The main difference is the location of the scissile phosphate group and the O3′ leaving group after cleavage: each group is displaced by ~1.3 Å from the position it occupied when the phosphodiester bond was intact (). Following cleavage, the phosphate group is hydrogen bonded with K83 (3.0 Å) (). The two Mg2+
ions are positioned similarly to the Ca2+
ions although they are slightly closer (3.6 Å as opposed to 4.2 Å). The smaller Mg2+
may shift the two metal sites closer together or a small shift in metal-ion coordination could be associated with activation of the enzyme, as suggested for the two Mg2+
ions in the RNase H–substrate complex (33
). Because the scissile phosphate group and the O3′ leaving group are involved in the metal ion coordination both before and after cleavage, the distance between the two metal ions is determined by the configuration of the active-site residues (E, D, Q or N) as well as the substrate.
Mg2+ at the metal m1 position is coordinated by the side chain oxygen atoms of D62 (Oδ2) and Q81 (Oε1), the main chain oxygen atom of V82 and one of the oxygen atoms of the cleaved phosphate group, all within a coordination distance of 1.9–2.1 Å. The coordination of the second Mg2+ at the m2 position is, however, somewhat looser: ranging from 2.0 Å (the side chain oxygen Oδ1 of D62), 2.15 Å (the O3′ leaving oxygen group), 2.3 Å (a water molecule), to ~2.6 Å (the oxygen atoms of the cleaved phosphate group). E18 is no longer coordinated to the metal at site m2 as in the Ca2+ complex. Overall, it appears that the metal at the m2 site is less tightly bound after the cleavage reaction.
K83 couples metal-ion coordination, sequence-specific recognition and DNA cleavage
The amino group of the side chain of K83, which is unlikely to be involved in the deprotonation of the nucleophilic water, is hydrogen bonded (3.2 Å) with the side chain oxygen Oε1 of Q81 (a metal coordinator) in the pre-reactive complex (). This hydrogen bond, however, is absent in the binary complex, where the amino group is re-oriented and hydrogen bonded (2.8 Å) instead with the carbonyl oxygen of Q93 (data not shown), a base recognition residue (). In the post-reactive complex, the amino group is within hydrogen bonding distance with three groups, the carbonyl oxygen of Q93 (2.85 Å), the side chain oxygen of Q81 (3.25 Å) and the scissile phosphate group (3.0 Å), after cleavage, in addition to its electrostatic interaction (). Thus, it appears that K83 couples metal-ion coordination and DNA cleavage with sequence-specific recognition, a linchpin model proposed recently for the function of the corresponding K79 in MutH (9