Download Characterization the binding of divalent metal and phosphate ions in

4. Discussion
4.1 The roles of divalent metal ions in the H-N-H motif in DNA hydrolysis
Divalent metal ions play important roles in DNA hydrolysis. The mostly widely
known examples are probably the requirement of Mg2+ for restriction enzymes in
cleavage of DNA substrates (Pingoud and Jeltsch 1997). Detailed structural analysis
of type II restriction endonucleases show that they catalyze the DNA cleavage via
similar two-metal-ion or three-metal-ion catalysis pathways (Kovall and Metthews
1999). Mg2+ ions play important catalytic roles in DNA hydrolysis catalyzed by
restriction enzymes that they may function as: general bases to activate the
nucleophilic water molecules for an in-line attack of the scissile phosphate; Lewis
acids to stabilize the negative-charged pentavalent phosphorous transition state; or
general acids that the Mg-bound water donating a proton to the hydroxyl leaving
group. As well as Mg2+ ions, divalent transition metal ions have also been identified
in the active sites of endonucleases, such as Zn2+ ions in P1 nuclease from Penicillium
citrinum (Romier et al. 1998) and Endonuclease IV from Escherichia coli (Hosfield et
al. 1999). In these zinc-dependent endonucleases, a three-metal-ion catalysis
pathway was proposed with zinc ions functioning either as general bases activating
nucleophilic water molecules or stabilizing scissile phosphate/hydroxyl leaving
groups.
In our previous study, we found that a water molecule was bound to the zinc ion in
the H-N-H motif of nuclease-ColE7 (Ko et al. 1999). In this study, we show that a
phosphate ion replaces the water molecule and forms a bridge between the zinc ion
and a histidine residue. The Mn2+ ion binds at the same site and coordinates to the
same residues as that of zinc ion. A similar result has been reported for Ni2+-bound
nuclease-ColE9/Im9 in which a phosphate ion is also bridged between the Ni2+ ion
and a histidine residue (Kleanthous et al. 1999). However, we did not find any Mg2+
binding sites either in apo-enzyme or zinc-holo-enzyme crystals soaked with Mg2+
ions. The original metal-binding site in the H-N-H motif of the apo-enzyme soaked
with Mg2+ ions was occupied with water molecules similar to that of EDTA-treated
crystals (data not shown), suggesting that the Mg2+ ion likely does not bind at the
center of the H-N-H motif in ColE7. This result was expected, since a magnesium
ion does not favor the binding site of a transition metal ion coordinated with three
histidine residues (Dudev and Lim 2001).
A previous structural comparison shows that the zinc ion in the H-N-H motif
matches well with the Mg2+ ion in active sites of I-PpoI and Serratia nuclease
(Kuhlmann et al. 1999). The Mg2+ in I-PpoI and Serratia nuclease are involved in
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stabilization of the phosphoanion transition state and in providing a proton to the 3’
oxygen leaving group. Therefore we suspect that the zinc ion located in the H-N-H
motif has a similar function for the stabilization of the phosphoanion transition state.
Because Mg2+ ion further enhanced the nuclease activity of the zinc-bound
holo-enzyme, it is possible that there is a second metal binding site for Mg2+ in
colicins but it is likely that Mg2+ is only bound upon DNA binding. A hypothesis
has been proposed that nuclease ColE9 hydrolyses DNA by two distinct catalytic
mechanisms in the presence of only Mg2+ or Ni2+ (Pommer et al. 2001; Walker et al.
2002). However, alkaline earth metal ions (Mg2+ in mM) and transition metal ions
are both present in cells and most likely ColE7 is a zinc-enzyme in vivo giving the
facts that Zn2+ binds colicins with highest affinity and Zn2+ is one of the most
abundant transition metal ions in cells.
Therefore, we propose here that
nuclease-ColE7 functions as a zinc-enzyme and Mg2+ ions are also involved in DNA
hydrolysis in vivo.
It has been shown before that the zinc binding to the apo-nuclease-ColE9 will
increase the proteolytic resistance and melting point of the enzyme; therefore, it was
conclude that the metal ion in the H-N-H motif serves a structural role. However,
our previous study indicates that the zinc ion in the H-N-H motif of ColE7 also
appears to be involved in the catalytic pathway (Ku et al. 2002). Firstly,
nuclease-ColE7 is bound to zinc ion in a one-to-one stoichiometry and this
holoenzyme is active for DNA hydrolysis. Removal of the zinc ion will result an
apo-enzyme with no DNase activity. Secondly, mutations in the vicinity of the
metal binding site impair DNase activity. Thirdly, although the apo-enzyme has no
DNase activity, it retains the ability of binding DNA. Since the zinc ion in the
H-N-H motif is not required for DNA binding, nut is required for DNA hydrolysis, so
it is likely that the zinc ion is involved in DNA hydrolysis.
4.2 His545 is likely the general base
In nuclease-ColE7, there are three histidine residues, His544, His569 and His573,
directly bound to the zinc ion. These three residues are not strictly conserved among
H-N-H proteins, but the residues that are aligned with these histidines are either polar
or charged and capable of binding to metal ions. Therefore, the residues aligned
with these histidines are likely to be responsible for metal ion binding. It is however
less clears what are the roles for the two most conserved residues, His545 and Asn560,
in the H-N-H motif. A structural comparison between the H-N-H motif and the
active sites in I-PpoI and Serratia nuclease sheds light on the possible functions for
His545 (Kleanthous et al. 1999).
The superimposition of the ββα-fold between
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nuclease-ColE7 and I-PpoI shows that His545 in ColE7 locates at a similar position
as His98 in I-PpoI (Figure 8B). His98 in I-PpoI serves as a general base to activate
a water molecule, which functions as a nucleophile to attack the scissile phosphate
group. In our structures, a phosphate ion is bound between His545 and a zinc ion
and the distance between His545 (ND1 atom) and phosphate oxygen is 2.6 Å.
Therefore it is very likely that His545 functions as a general base. This could
explain the strict requirement for a histidine at this position in the entire H-N-H
family proteins.
4.3 Asn560 likely stabilizes the loop region of the N-H-N motif
The second most conserved residue in H-N-H motif is Asn560, which is almost
always strictly conserved in H-N-H motif with only a few exceptions. The side
chain of Asn560 makes a hydrogen bond network with the main-chain atoms in the
long loop region with the amide group being fully occupied (Figure 17). The ND2
atom makes two hydrogen bonds with the carbonyl groups of Gly554 and Lys547 and
the OD1 atom also makes two hydrogen bonds with the amino groups of Lys547 and
Glu546. It appears that Asn560 has no unoccupied positions in polar atoms to
participate in hydrolysis. Therefore, it is likely that Asn560 only plays a structural
role to fix the backbone structure in the loop region so that the general base residue
His545, which hydrogen bonds to the carbonyl group of Val555, is appropriately held.
4.4 A structural model for nuclease-ColE7 bound to DNA
The crystal structure of I-PpoI in complex with DNA demonstrates at the atomic
level how this enzyme works (Flick et al. 1998; Galburt et al. 1999; Galburt et al.
2000). I-PpoI has an elongated fold with a concave surface to bind and cleave DNA
at the minor groove of its homing site. The two active sites in the dimeric I-Ppo I
are about 20 Å apart, therefore in order to place the two farthest away scissile
phosphate groups near the active sites, the DNA is bent by approximately 50o.
A structural model for nuclease-ColE7 bound with DNA was constructed based
on the crystal structure of the I-PpoI/DNA complex (Figure 18). The H-N-H motif
in nuclease-ColE7 was superimposed with the two β-strands and one α-helix in the
active site of I-PpoI with an rms difference of only 1.2 Å for the main-chain atoms.
Without further adjustment, the nuclease-ColE7 appears to bind DNA snugly. The
concave surface of nuclease-ColE7 faces the DNA backbone with the zinc ion
positioned close to the phosphate backbone at the minor groove, appropriate for its
role in stabilizing the phosphoanion transition state. The α-helix, α2, binds DNA at
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the neighboring major groove, and there are indeed several basic residues (R496,
K497, K498, K505) located in this helix. The putative nuclease active site in this
model did not blocked by Im7, the Im7 original binding site was overlaps with DNA,
so this model supports the thory that Im7 block the DNA binding site in ColE7,
resulting in the inhibition of its nuclease activity.
4.5 The possible mechanism for nuclease-ColE7 hydrolyze in hydrolyzing DNA
Enzymes have long been appreciated for their ability to efficiently and specifically
catalyze chemical transformations. To efficiently catalyze the cleavage of phosphate
esters, certain chemical elements are required. Many nucleases, including restriction
and homing endonucleases, exonucleases, and some of ribozymes, use the bound
metal ions as cofactors. Endonucleases utilize one, two, or even three bound metals
have been observed and studied extensively. Metal ions can act as Lewis acids by
lowering the pKa of their directly coordinated water molecules. A resulting
hydroxide may then serve as either a nucleophile or a general base. Alternatively, an
acidic, metal-bound water molecule can efficiently protonate the leaving group.
Perhaps most importantly, the positive charge of the divalent metal ion can stabilize
the -2 charge that accumulates at the phosphoanion transition state relative to the -1
charge of the ground state. All three of these functions could lower the free energy
of the transition state and accelerate the reaction.
The catalytic mechansisms for several homing endonucleases have been studied
extensively and can be used to serve as examples to show how divalent metal ions
participate in DNA hydrolysis. The homing endonuclease I-CreI belongs to the
LAGLIDADG subgroup of homing endonucleases which comprise the largest homing
endonuclease family with many more than 200 known members. Based on the
high-resolution cocrystal structure between I-CreI and its DNA homing site, the
I-CreI active site appears to conform to a canonical two-metal mechanism. One metal
positions and activates the nucleophilic water, and the second metal stabilizes both the
transition state phosphoanion and the oxygen leaving group.
The His-Cys box homing endonuclease family is defined by two clusters of
histidine and cysteine that form structural zinc binding sites. Zinc is required for
protein folding, but does not directly play a role in catalysis. The His-Cys box
homing endonuclease I-PpoI uses a single-metal ion mechanism in which a
magnesium ion accelerates the reaction by stabilizing the phosphoanion transition
state, positioning and activating a water molecule to donate a proton to the leaving
group oxygen, and introducing strain in the substrate complex that is relieved in the
product complex (Figure 19). The bound metal is coordinated by Asp119, the
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leaving group oxygen, a nonbridging oxygen on the scissile phosphate, and three
water molecules in the substrate complex. In the product complex, the now terminal
5′-phosphate moves away from the metal and toward Arg61. The leaving group is
stabilized by the metal ion, and a fourth water molecule replaces the nonbridging
oxygen as the sixth metal ligand. The nucleophilic water has been observed in the
crystal structures and is positioned nicely for in-line attack. His98 is the general
base, and a hydrogen bond is observed between the nucleophile and δN of His98.
The backbone carbonyl of a zinc-coordinating cysteine (Cys105) stabilizes the correct
tautomer of His98 by hydrogen bonding to the εNH group.
Base on the superposition of the H-N-H motif in nuclease-ColE7 and the active
site in I-PpoI, we purpose a possible mechanism of DNA hydrolysis catalyzed by
nuclease-ColE7 (Figure 20). In the crystal structure of the nuclease-ColE7/Im7, the
bound zinc metal ion is coordinated by His544, His569, His573, and the scissile
phosphate.
Threrefor the zinc ion likely involved in stabilization of the
phosphoanion transition state. His545 likely functions as a general base, activating a
water molecule for the nucleophilic attack to the phosphate. A water molecule,
likely near the His569, functioning as a general acid donating a proton to the leaving
group oxygen.
However, one puzzling question remained: what is the general acid in this DNA
hydrolysis reaction? In I-PpoI or Serratia nuclease, the magnesium-bound water
molecule serves as the general acid to provide a proton for the 3’ oxygen leaving
group. In the nickel-bound nuclease-ColE9 structure, the Ni2+ ion was bound with
two histidine residues and one water molecule, therefore, it was proposed that this
nickel-bound water molecule functions as a general acid (Pommer et al. 2001; Walker
et al. 2002). However, in our zinc-bound holoenzyme, the zinc ion was in a
tetrahedaral not an octahedral geometry, and Zn2+ was not bound with a water
molecule. We did not find any residue surrounding the zinc ion that seems likely to
function as a general acid. One possibility is that the zinc ion acquires another water
molecule and coordinates to six ligands after it is bound to DNA. Or it is likely that
a magnesium ion also participates in this reaction and that the magnesium-bound
water molecule donates a proton to the 3’ oxygen leaving group. We are eagerly
pursuing the crystal structures of the nuclease-ColE7/DNA complex, which will
reveal unambiguously the arrangement of substrate binding and cleavage catalyzed by
H-N-H proteins.
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