(Received for publication, October 5, 1995; and in revised form, December 19, 1995)
From the
According to the crystal structure of the specific EcoRVDNA complex, not only the functional groups of the
nucleobases but also the phosphate groups of the DNA backbone are
contacted by the enzyme. To examine the contribution of backbone
contacts to substrate recognition and catalysis by EcoRV, we
exchanged 12 amino acids residues located close to phosphate groups by
site-directed mutagenesis. We purified the resulting EcoRV
mutants and characterized them with respect to their DNA binding and
cleavage activity. According to our steady state kinetic analysis,
there are strong interactions between three basic amino acid residues
(Lys-119, Arg-140, and Arg-226) and the phosphate backbone that support
specific binding presumably by inducing and maintaining the kinked
conformation of the DNA observed in the specific EcoRV
DNA complex. These contacts are important in both
the ground state and the transition state. Other, uncharged residues
(Thr-93 and Ser-112), which could be involved in hydrogen bonds to the
phosphate groups, are needed primarily to stabilize the transition
state. An especially important amino acid residue is Thr-37, which
seems to couple recognition to catalysis by indirect readout.
The specific interaction of proteins with DNA is of fundamental importance in biology. Much effort, therefore, has been undertaken to understand the molecular basis of the underlying recognition process. It has been suggested that specificity is due to specific hydrogen bonds between the protein and the edges of the bases of the DNA (direct readout)(1) . Such contacts are indeed observed in nearly every structure of specific protein-DNA complexes determined so far. In the first co-crystal structure of the trp repressor and its operator DNA, however, no direct contacts to the bases were seen that could account for the observed specificity of DNA binding; it was argued, therefore, that in this case, specific recognition of DNA is primarily due to contacts to the phosphate groups of the DNA backbone (indirect readout)(2) . This means that during interaction with DNA, the protein recognizes a specific sequence-dependent conformation of the phosphodiester backbone in addition to functional groups of the bases. This concept was initially met with some reservation, but it has proven to be very fruitful, although it was recently demonstrated that the specific interaction between the trp repressor and its operator is not only due to phosphate contacts but also due to water-mediated base contacts as well as DNA-induced tetramerization of the protein on the DNA(3) . From comparisons of co-crystal structures of specific and nonspecific complexes of DNA binding proteins with DNA, it became evident that specific recognition of DNA by proteins is mediated by direct and indirect readout also in other systems (e.g. glucocorticoid receptor(4) ; EcoRV(5) ); in general, in the specific complexes, much more interactions are observed between the protein and the bases but also between the protein and the phosphate backbone of the DNA than in the nonspecific complexes.
It is difficult to assess the relative
contributions of both mechanisms of specific recognition because in
virtually all complexes between specific DNA binding proteins and their
cognate DNA, direct and indirect readout is the result of an
interconnected network of hydrogen bonds to functional groups of the
bases exposed in the major and minor grooves and hydrogen bonds or
ionic interactions to the phosphate groups of the DNA. The crystal
structure analyses of restriction enzymeDNA complexes provide
beautiful examples that illustrate the complexity of the network
characteristic for the protein-DNA interfaces, as demonstrated for EcoRI(6, 7) , EcoRV(5, 8) , PvuII(9) , and BamHI (10) . In the case of EcoRV, there are
various reasons to assume that the indirect readout contributes to the
recognition process. In contrast with the co-crystal structures of the
other restriction enzymes, where direct or water-mediated hydrogen
bonds are observed to every base pair of the respective recognition
sequences, in the specific EcoRV
DNA co-crystal
structure, no hydrogen bonds to the bases of the two inner AT base
pairs of the recognition sequence GAT
ATC (the arrow denotes the
position of phosphodiester bond cleavage) are seen. Furthermore, among
the four co-crystal structures obtained so far for restriction enzymes,
the EcoRV
DNA structure is unique in having a highly
distorted DNA with a sharp central kink of approximately
50°(5, 11, 12) , which renders the inner
two base pairs inacessible to the protein. Recognition of these two
base pairs, therefore, cannot be direct. On the other hand, there are a
multitude of amino acid residues contacting the phosphate groups of the
DNA both within and outside of the recognition sequence (5) (see Fig. 1), which might compensate for the lack of
base contacts.
Figure 1:
Amino acid residues contacting the
phosphate backbone of the DNA. In a, a schematic
representation of backbone contacts between EcoRV and one
strand of the DNA are given. Only residues that harbor a hydrogen bond
donor in a distance <0.4 nm from one of the nonbridging oxygen atoms
of the phosphate groups in at least one of the EcoRVDNA
co-crystal structures are shown. These residues and the contacted DNA
strand are highlighted in the structural model of the specific
undecamer complex (Brookhaven data bank entry 1RVA) shown in b. O
refers to side chain oxygens of Ser and Thr
residues, O
refers to the side chain oxygen of a Tyr
residue, and N
and N
refer to the terminal
nitrogen atoms of Arg and Lys residues,
respectively.
One strategy applied to examine the indirect readout
of restriction enzymes has been to use modified oligonucleotides as
substrates, e.g. phosphorothioates (EcoRI(13, 14) , EcoRV())
or S-methylphosphorothioates (TaqI(16) ).
Here we present a complementary approach focussing on the contribution
of the protein to the indirect readout by a systematic mutational
analysis of amino acid residues, which in the crystal structure are
located sufficiently close to the phosphodiester backbone to be
considered candidates for indirect readout. We will present evidence
that the catalytic efficiency of the restriction enzyme EcoRV
depends on both modes of specific recognition.
For K and k
determination, the initial
velocities were calculated from the linear part of the progress curves
obtained with six or more different substrate concentrations. The K
and k
values were
obtained by a best fit to the cleavage data using the computer program
ENZFITTER (Biosoft(TM), Cambridge, UK). Substrate concentrations
were chosen to cover at least a range from 0.2 to 5 K
.
Figure 2:
Cleavage of linearized pATRV by the S112A
mutant (a) and wild-type EcoRV (b) in the presence
MnCl. The DNA concentration used was 21 nM; the
enzyme concentration was 50 nM. The locations of substrate and
product bands resulting from cleavage at the canonical EcoRV
sites are indicated (cf. legend to Fig. 3); all other
bands are due to star activity.
Figure 3:
Cleavage of linearized pATRV by the R226A
mutant (a) and wild-type EcoRV (b) in the presence of
MgCl. The DNA concentration used was 21 nM, and
the enzyme concentrations were 10 and 1 nM, respectively.
There are two EcoRV sites on the 3678-bp plasmid: cleaving
first at the . . . GCGGGATATCGTCC . . . site gives a 2665- and a
1013-bp fragment, whereas cleaving first at the . . . GAAAGATATCAAAA .
. . site yields a 2261- and a 1417-bp fragment; complete digestion
results in three products of 1417, 1248, and 1013
bp.
It is known that the DNA cleavage rate of EcoRV is influenced by sequences flanking the recognition sequence(27) . The use of a plasmid substrate with two EcoRV sites embedded in very different sequence surroundings offers the possibility to screen the mutants for an enhanced sensitivity toward flanking sequences. Whereas in cleavage buffer the wild-type enzyme cleaves both sites with similar rates, three EcoRV variants, namely R226A, R140A, and T93A, show a preference for one site (Table 2, Fig. 3), all three mutants prefer the EcoRV site with AT rich flanking sequences.
Figure 4: Michaelis-Menten diagrams for the cleavage of d(pGATCGACGATATCGTCGATC) with wild-type EcoRV (a) and the mutant R226A (b). The values for the initial rates are averages from at least three independent experiments; they are accurate within ± 30%.
Depending on their K and k
values, it is possible to divide the EcoRV mutants into three groups (Table 3).
Group I
consists of mutants with a similar k/K
value as the wild-type
enzyme: S41A, T94A, Y95F, T111A, R221A, S223A.
Group II consists of
mutants with lower k/K
values due to an increase in K
: K119A,
R140A, R226A. The k
/K
values of these mutants are reduced by 2-3 orders of
magnitude compared with the wild-type enzyme, and this group,
therefore, comprises the EcoRV variants with the lowest
activities.
Group III consists of mutants with lower k/K
values due to a
decrease in k
: T93A and S112A. The k
/K
values of these mutants
are by more than 1 order of magnitude lower than that of the wild-type
enzyme.
The only EcoRV variant that could not be classified
according to the above scheme is the T37A mutant. Due to its low
activity, DNA cleavage by T37A is only measurable at equimolar
concentrations of enzyme and substrate, i.e. under single
turnover conditions. In good agreement with the results of the plasmid
DNA cleavage experiments (Table 2), the single turnover cleavage
rate constant k for oligodeoxynucleotide DNA cleavage by this
mutant is 700 times lower than the k of the
wild-type enzyme (Table 3). As the K
value
measured for T37A (see below) is well below the substrate concentration
that was employed in the single turnover experiments (0.5
µM), the enzyme is likely to be saturated with substrate.
Hence, it is reasonable to compare the k value of the mutant
with the k
value of the wild-type enzyme. Thr-37
is the only amino acid residue involved in a phosphate contact that has
an impact on both specific binding (K
) and
catalysis (k), although the effect on catalysis is much more
pronounced.
Figure 5:
Binding of wild-type EcoRV (a)
and the mutant R226A (b) to a 382-bp DNA fragment in the
presence of CaCl. The binding was studied with 20 pM (a) and 1 nM (b)
P-labeled
DNA and EcoRV at the concentrations indicated. The bands
corresponding to the specific complex and the free DNA are
indicated.
For the wild type enzyme and
most of the mutants, the K values measured in the
presence of Ca
(which permits cleavage, but supports
binding) are 1 order of magnitude lower than the K
values measured in the presence of Mg
. In a
double logarithmic plot of K
versus
K
, most mutants are well represented by the regression
line (Fig. 6). Exceptions are the mutants T93A and S112A; their K
values are more than 1 order of magnitude higher
than the corresponding K
values. As in the two
sets of experiments (binding versus cleavage) only the
divalent cation used was different; this finding may suggest that in
the presence of specific DNA, these two mutants have a lower affinity
for Ca
than for Mg
compared with
the wild-type enzyme and the other mutants.
Figure 6:
Double logarithmic plot of Kversus K
for wild-type EcoRV and all EcoRV mutants. The data
were taken from Table 3. The slope of 1 of the regression line
suggests that wild-type EcoRV and the various mutants, with
exception of S112A and T93A, differ by a constant energy increment
(
G = RT ln (K
/K
)) of
5.7 kJ or 1.4 kcal in their DNA binding affinity in the presence of
Mg
and Ca
,
respectively.
Restriction endonucleases recognize their cleavage sites on
double-stranded DNA with remarkable high specificity by forming
contacts both to the bases and to the backbone of the
DNA(28, 29) . As the essential role of the base
contacts had been established before (21, 18) , it has
been the aim of the work presented here to define the importance of
contacts between amino acid residues and phosphate groups for the
mechanism of DNA recognition by the restriction endonuclease EcoRV. For this purpose, we have exchanged all amino acid
residues that are located in proximity to the phosphodiester backbone
of the DNA in the co-crystal structures of EcoRVDNA
complexes(5, 8) . The resulting EcoRV
variants were analyzed in terms of DNA binding and cleavage activity.
According to our experimental results, the amino acid residues Thr-37,
Arg-226, Lys-119, Arg-140, Thr-93, and Ser-112 (arranged in the order
of decreasing importance for catalysis) are of major importance for EcoRV activity. They are located close to the phosphates
+2, -5, -2, +3, +1, and -1,
respectively (Fig. 1a). This finding is in agreement
with the results of a complementary analysis in which phosphate
contacts of EcoRV were probed with phosphorothioate containing
oligonucleotides; it was shown that all phosphates demonstrated by our
study to be subject to indirect readout show effects upon modification
(the position +5 was not subject of this study).
The various mutants can be classified according to whether
they are impaired in K and/or k
. In the subsequent discussion this
classification will be used.
Interestingly, some of the mutants
(T94A, Y95F, and R221A), which have a similar k/K
value as the wild-type
enzyme, have a 2-fold higher k
and K
value (group I* in Table 3). Moreover,
their K
is also higher than that of the wild-type
enzyme. These results may be interpreted in terms of small differential
effects on substrate binding and transition state stabilization; it
appears that these residues are involved in contacts that stabilize the
ground state of the enzyme-substrate complex, not, however, the
transition state (Fig. 7).
Figure 7:
Schematic free energy profiles to
illustrate the classification of EcoRV mutants in groups I*, II, and
III. The relative energy levels of the various states of the wild-type
enzyme and the mutants encountered during enzymatic turnover (E + S ES
ES
E + P) are represented by solid and dashed
lines, respectively. The K
value is
represented by the energy level of the ground state complex (ES), the k
value by the energy
difference of the ground state and the transition state complex (ES
). Note that the energy levels are not drawn to
scale, neither within nor among the groups.
We suggest
that the main function of the residues Arg-226, Lys-119, and Arg-140 is
to stabilize the distorted conformation of the DNA in the specific EcoRVDNA complex. The results obtained for the K119A and
R140A mutants can be rationalized by a comparison of the co-crystal
structures of EcoRV with specific and nonspecific DNA. Upon
formation of the specific complex, there is a significant movement of
the residues Lys-119 and Arg-140 with respect to the DNA that shortens
the distance between the interacting partners (Fig. 8, Table 1). Thus, weak ionic interactions with nonspecific DNA
become considerably stronger in the complex with the specific substrate
leading to a gain in binding energy at the transition from the
nonspecific to the specific binding mode. The distances between
Lys-119, Arg-140, and the phosphate groups -2 and +3,
respectively, are not the only ones that are altered in the co-crystal
structures with nonspecific and specific DNA (Table 1), but we
suppose that the changes in positions of Lys-119 and Arg-140 are the
most important ones because the interacting partners are charged.
Figure 8:
Details of the superimposed
EcoRVDNA co-crystal structures with specific (Brookhaven data
bank entry 1RVA) and nonspecific DNA (dashed lines; Brookhaven
data bank entry 2RVE). The two structures are centered on the phosphate
atom of the phosphate group contacted by Lys-119 of subunit A (a) and Arg-140 of subunit B (b). The distances (in
nm) between functional groups of the amino acid side chains and
nonbridging oxygen atoms of the respective phosphate group are
indicated.
Whereas the decrease in apparent binding energy that accompanies the
substitution of Lys-119 (6.7 kJ/mol or 1.6 kcal/mol) and Arg-140 to
alanine (4.0 kJ/mol or 0.95 kcal/mol), respectively, are roughly
compatible with the deletion of a single phosphate contact, the energy
penalty associated with the exchange at position 226 (8.2 kJ/mol or 2.0
kcal/mol) is significantly higher. The large effect observed with the
R226A mutant is particularly noteworthy as Arg-226, which is in a
disordered region in the co-crystal structure of the unspecific EcoRVDNA complex, contacts the DNA backbone two residues
outside of the recognition sequence. This implies that Arg-226 must
have an additional role. It has been suggested that this amino acid
residue apart from promoting specific binding is responsible for the
stabilization of a loop between
helices D and E encompassing
residues 221-228 (8) (cf.Fig. 1b). The importance of the C terminus of EcoRV for catalysis was demonstrated previously with a mutant
whose C-terminal end, starting from position 216, had been deleted.
This mutant showed no activity in cleavage assays with
-DNA(21) . Furthermore, the W219C mutant was shown to be
very defective in DNA binding in the presence of
Mg
(20) .
In comparison with the wild-type enzyme, two mutants of group II (R140A, R226A) show an enhanced selectivity toward different flanking sequences (Table 2, Fig. 3). Flanking sequences are likely to influence the structure and dynamics of the DNA, both of which are important in the recognition process, especially for the propensity of the recognition sequence to be bent in a unique way. The EcoRV restriction endonuclease distorts the recognition sequence GATATC during the recognition process by introducing a sharp central kink(5) . It appears, as if reducing the ability of EcoRV to deform the DNA, for example through mutation of an amino acid residue that stabilizes the kinked conformation, leads to an increased sensitivity of EcoRV to flanking sequence effects. This consideration may explain the preferences of some phosphate contact mutants for EcoRV sites with AT-rich flanking sequences because these sequences may facilitate the distortion of the cognate DNA during the recognition process.
A remarkable
property of these mutants is a pronounced metal ion effect on the
binding of DNA. In the presence of Ca, T93A and S112A
bind to the DNA by more than 1 order of magnitude more weakly than in
the presence of Mg
, whereas the wild-type enzyme and
all other mutants bind to DNA by 1 order of magnitude more strongly in
the presence Ca
than in the presence of
Mg
(Table 3, Fig. 6). These results
suggest that the residues Ser-112 and Thr-93 participate in the correct
assembly of binding sites for divalent metal ions important for
specific binding. In agreement with these results, metal ion binding
sites of EcoRV have been mapped to be in the catalytic center
of the enzyme (5, 8) and at a site remote from the
cleavage site at phosphate -2(20) . Whereas Thr-93 is in
close proximity to the catalytic center and hence may affect this metal
binding site, the main chain carbonyl group of Ser-112 is one possible
ligand for the second metal binding site. This conclusion is supported
by our finding that the replacement of Ser-112 (and of Thr-111) by
alanine leads to an EcoRV variant with a relaxed specificity
in the presence of Mn
.
Figure 9:
Detail of the EcoRVDNA co-crystal
structure with specific DNA (Brookhaven data bank entry 1RVA). The
connections between Thr37 and other functional important regions of EcoRV are depicted. The thin line represents helix B,
distances (in nm) are marked with dashed lines. The capital letters in parenthesis refer to the different EcoRV subunits A and B,
respectively.
It is presumably no coincidence that Thr-37 as
well as Thr-93 and Ser-112, which when substituted by alanine produce
the largest k effects, also show the largest
movements in a comparison of the enzyme-substrate and enzyme-product
complexes ( Table 1and Table 3).
In summary, the
restriction enzyme EcoRV uses both direct and indirect readout
mechanisms to achieve its high accuracy of recognition. The primary
function of basic amino acid side chains contacting the phosphate
backbone of the DNA is to stabilize the kinked DNA conformation in the
specific EcoRVDNA complex. Other, uncharged residues
help to assemble the catalytic center in the transition state. An
especially important residue, Thr-37, is most probably responsible for
the coupling of specific recognition and catalysis.
The effects seen with mutants of amino acid residues in close vicinity to the phosphate backbone are in general not as severe as observed with mutants of the recognition loop of EcoRV (21) but together may contribute to a considerable extent to the specificity of the recognition process and to the catalytic efficiency.