(Received for publication, April 18, 1995; and in revised form, June 21, 1995)
From the
The Escherichia coli RNase HI variant with the
Lys
Ala mutation is purified in two forms, as
nicked and intact proteins. The nicked K86A protein, in which the
N-fragment (Met
-Lys
) and the C-fragment
(Arg
-Val
) remain associated, is
enzymatically active. These N- and C-fragments were isolated and
examined for reassociation. These peptides did not associate to form
the nicked K86A protein at pH 3.0 in the absence of salt, but were
associated, with a yield of 30-80%, when the pH was raised to 5.5
or when salt was added. Measurements of the CD spectra show that the
-helices are partially formed in the N-fragment at pH 3.0 in the
absence of salt and are almost fully formed either at pH 5.5 or at pH
3.0 in the presence of 0.15 M NaCl. In contrast, the
C-fragment remains almost fully disordered under these conditions. The
N-fragment with this high (native-like) helicity shows the
characteristics of a molten globule with respect to the content of the
secondary and tertiary structures, the ability to bind a fluorescent
probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the
thermal transition. These results suggest that the N-fragment contains
an initial folding site, probably the
I-helix, and the completion
of the folding in this site provides a surface that facilitates the
folding of the C-fragment. This folding process may represent that of
the intact RNase HI molecule.
Escherichia coli ribonuclease HI, which
endonucleolytically hydrolyzes only the RNA strand of a DNA/RNA hybrid,
is composed of a single polypeptide chain with 155 amino acid residues
(Kanaya and Crouch, 1983). Crystallographic studies revealed that this
protein is of the +
structure, and is composed of a
five-stranded
-sheet and five
-helices (Katayanagi et
al., 1990, 1992; Yang et al., 1990). A similar folding
topology has been weakly observed in other functionally unrelated
proteins, such as hexokinase and glycerol kinase, and it is therefore
defined as an RNase H-like fold by Holm and Sander(1994). Recently, the
crystal structures of the catalytic domain of human immunodeficiency
virus type-1 integrase (Dyda et al., 1994) and the Holliday
junction resolving enzyme RuvC from E. coli (Ariyoshi et
al., 1994) were shown to have this RNase H-like fold. These
proteins are functionally unrelated to E. coli RNase HI (EC
3.1.26.4) and have little sequence similarity. Dyda et
al.(1994) and Yang and Steitz(1995) have suggested that this
folding topology represents the structures of similar types of enzymes
that catalyze polynucleotidyl transfers.
We have previously
introduced Ala-scanning mutagenesis into the basic protrusion of E.
coli RNase HI to analyze the role of this region in substrate
binding (Kanaya et al., 1991). Of the 16 mutant proteins in
which the amino acid residues from His to
Asn
, except for Ala
, were individually
replaced by Ala, only the K86A protein, with the Lys
Ala mutation, was purified in two forms. One is the intact
protein, and the other is the nicked protein. In the nicked protein,
the peptide bond between Lys
and Arg
, which
is located nearly in the middle of the protein molecule, is cleaved,
but the resultant N-fragment (Met
-Lys
)
and the C-fragment (Arg
-Val
) remain
associated (Kanaya et al., 1991). It remains to be determined
which protease specifically cleaves this peptide bond, and why the
Lys
Ala mutation makes this peptide bond susceptible
to proteolysis. The crystal structure of E. coli RNase HI, in
which the N-terminal segment (1-87) is distinguished from the
C-terminal segment (88-155), is schematically shown in Fig. 1. The N-terminal segment forms the initial four
-strands (
A-
D) and three
-helices
(
I-
III), and the C-terminal segment forms the remaining
-strand (
E) and two
-helices (
IV,
V). Studies
of the dissociation and association (complementation) of the N- and
C-fragments, as well as their structural analyses, will be of special
interest, because these studies should provide valuable information on
the folding process of E. coli RNase HI, which may represent
those of proteins with the RNase H-like fold.
Figure 1:
Main chain folding of E. coli RNase HI. The crystal structure of E. coli RNase HI,
determined by Katayanagi et al.(1990, 1992), was drawn as a
ribbon model with the program MOLSCRIPT (Kraulis, 1991). The position
of the peptide bond between Lys and Arg
,
which is cleaved by a protease when Lys
is replaced by
Ala, and the side chain of Lys
are indicated. The solid and shaded regions correspond to the N- and
C-fragments. The
III-helix and the long loop between the
III-
and
IV-helices form the basic protrusion. This crystal structure
of E. coli RNase HI is deposited in the Protein Data Bank,
Brookhaven National Laboratory, with the accession number
2RN2.
Generally, a folding intermediate of a protein is very unstable, because of the very rapid completion of the following folding process to the native state. Folding intermediates have been analyzed by NMR measurements of hydrogen exchange (Matouschek et al., 1990; Miranker et al., 1991, 1993; Neri et al., 1992; Jennings and Wright, 1993) or by CD (Shortle and Meeker, 1989; Griko et al., 1994). Fragmentation and complementation experiments are expected to be an alternative strategy to analyze folding intermediates of proteins. If a protein molecule were cut into a few pieces that could be reassembled to form the nicked protein, structural analyses of the individual peptide fragments would provide information on the structure of the folding intermediates of the intact protein molecules. Since it was first reported by Richards and Vithayathil (1959) for RNase A, the complementation of peptide fragments has been reported for various proteins, such as staphylococcal nuclease (Taniuchi and Anfinsen, 1971), barnase (Sancho and Fersht, 1992; Kippen et al., 1994), adenylate kinase (Girons et al., 1987), chymotrypsin inhibitor-2 (Gay and Fersht, 1994; Gay et al., 1994a, 1994b), bovine pancreatic trypsin inhibitor (Oas and Kim, 1988; Staley and Kim, 1990), E. colitrp repressor (Tasayco and Carey, 1992), and isoleucine-tRNA synthetase (Shiba and Schimmel, 1992). Such peptide fragments were created by chemical synthesis, proteolysis and chemical cleavage of the proteins, or genetic methods. The structures of various peptide fragments (Bierzynski et al., 1982; Osterhout et al., 1989; Fontana, 1990; Beals et al., 1991; Chaffotte et al., 1991; Dyson et al., 1992; Sancho et al., 1992; Waltho et al., 1993;), including a dissected protein molecule (Peng and Kim, 1994), have also been analyzed, assuming that a peptide containing an initiation site for protein folding spontaneously folds into a conformation that is similar to that of a folding intermediate of the intact protein molecule.
In
this study, we present thermal, chemical, and acid denaturations of the
nicked K86A protein, structural analyses of the N- and C-fragments, and
reconstitution of the nicked protein from the N- and C-fragments. We
propose that the initial folding site in E. coli RNase HI
resides in its N-terminal half region and that the formation of an
-helix in this region facilitates the following folding process of
the C-terminal half region.
The K86A protein was purified as a mixture
of the intact and nicked forms by the standard purification procedure
(Kanaya et al., 1989). The intact and nicked K86A proteins
were separated by cation-exchange HPLC on an Asahipak ES502C column
(7.5 100 mm) from Asahi Chemical Industries Co., Ltd. Elution
was performed by a linear gradient of 0.015-0.10 M
Na
SO
in 20 mM sodium phosphate buffer
(pH 6.5) over 30 min with a flow rate of 1.0 ml/min. Proteins were
detected by a variable wavelength UV detector at 280 nm. The retention
times of the intact and nicked K86A proteins were 23 and 26 min,
respectively. The cellular production level of the intact K86A protein
was 30-50 mg/liter, as judged by the intensity of the band
visualized by Coomassie Brilliant Blue staining of the SDS protein gel.
SDS-polyacrylamide gel electrophoresis was carried out on a 15%
polyacrylamide gel, as described by Laemmli(1970). The intact and
nicked K86A proteins were individually purified with yields of
20-40%.
The N-fragment
(Met-Lys
) and the C-fragment
(Arg
-Val
), which remain associated in
the nicked K86A protein, were separated by reverse-phase HPLC on an
Aquapore RP-300 column (4.6
250 mm) from Brownlee Laboratories.
Elution was performed by a linear gradient of 10-60% (v/v)
acetonitrile with 0.1-0.05% trifluoroacetic acid, over 25 min
with a flow rate of 1.0 ml/min. The retention times of the N-fragment,
the intact K86A protein, and the C-fragment were 18.1, 23.4, and 24.7
min, respectively.
The reversibility of the denaturation of the nicked protein in GdnHCl was examined by the following procedures. The protein was dissolved to a concentration of 4-5 mg/ml in 20 mM sodium acetate buffer (pH 5.5) containing 3 M GdnHCl, and then diluted by 20-fold with 10 mM Gly-HCl buffer (pH 3.0), 10 mM sodium acetate buffer (pH 5.5), and 10 mM Tris-HCl buffers (pH 7.8 and 9.4). These solutions were further diluted by 10-fold with 20 mM sodium phosphate buffer (pH 6.5) and were subjected to cation-exchange HPLC using an ES502C column to determine the amount of the reconstituted protein. This second dilution with 20 mM sodium phosphate buffer (pH 6.5) did not seriously affect the yield of the reconstitution, probably because the concentrations of the N- and C-fragments were too low to permit effective fragment association.
Thermal and GdnHCl denaturation curves, as well as the
parameters characterizing these denaturations, such as the midpoint of
the thermal transition (T), the midpoint
of the transition in the GdnHCl concentration ([D]),
the free energy change in H
O
(
G[H
O]), and the measurement of the
dependence of
G on the GdnHCl concentration (m),
were determined as described previously (Kimura et al., 1992),
by monitoring the change in the CD value at 220 nm.
Figure 2: CD spectra of the wild-type and K86A proteins. The near-UV CD spectra of the wild-type (thinline), intact K86A (brokenline), and nicked K86A (thickline) proteins are shown. These spectra were measured at pH 5.5 in the absence of GdnHCl, as described under ``Experimental Procedures.''
Figure 3:
Elution profile of the nicked protein from
a Sephadex G-75 column. The nicked K86A protein was applied to a
Sephadex G-75 column (0.6 50 cm), which was previously
equilibrated with 10 mM sodium acetate buffer (pH 5.5)
containing 3 M GdnHCl. The column was eluted with the same
buffer at a flow rate of 7 ml/h. Fractions (300 µl) were collected,
and an aliquot of each fraction was subjected to reverse-phase HPLC to
determine the concentrations of the N-fragment (
) and C-fragment
(
) simultaneously. Arrows indicate the positions at
which blue dextran (1), wild-type RNase HI (2), and
NaCl (3) eluted from the column.
The GdnHCl denaturation curves of the nicked and intact K86A
proteins are shown in Fig. 4. The replacement of Lys by Ala does not seriously affect the stability of the protein.
However, the introduction of the nick into this mutant protein
dramatically destabilizes the protein, by 1.07 M in
[D] (Table 2). The fragment dissociation might
be the reason for the dramatic decrease in the stability of the nicked
protein.
Figure 4:
GdnHCl-induced denaturation curves of the
intact and nicked K86A proteins. The apparent fraction of unfolded
protein, which was calculated as described previously (Kimura et
al., 1992), is shown as a function of GdnHCl concentration. ,
intact K86A protein;
, nicked K86A protein. GdnHCl denaturation
curves were determined at pH 5.5 and 25 °C by monitoring the change
in the CD value at 220 nm.
Figure 5:
CD spectra of the thermally denatured
nicked protein. The far-UV (a) and near-UV (b) CD
spectra of the nicked K86A protein in the native (thinline), D (thickline), and
D
` (brokenline) states are shown. The CD
spectra of the nicked protein in the native and D
` states
were measured at pH 3.0 and 10 °C, and that in the D
state was measured at pH 3.0 and 55
°C.
The far- and near-UV CD
spectra of the nicked protein in the D state were identical
with those of the wild-type and intact K86A proteins in the D
state. In contrast to the wild-type and intact K86A proteins,
however, the thermal denaturation of the nicked protein was not
reversible. The far- and near-UV CD spectra of the nicked protein,
which was thermally denatured at 55 °C and then refolded by
lowering the temperature to 10 °C, were similar to those of the
nicked protein in the D
state (Fig. 5). The
conformation of this nicked protein is therefore designated as the
D
` state. The far-UV CD spectrum of the nicked protein in
the D
` state exhibited a trough with a minimum
[
] value of -13,500 at 202 nm, which is
accompanied by a shoulder with a [
] value of
-6,500 at 220 nm (Fig. 5a). Gel filtration of the
nicked protein in the D
` state with Sephadex G-75 indicates
that the N- and C-fragments are dissociated from each other at pH 3.0
upon thermal denaturation and remained in monomeric forms even when the
temperature was lowered to 10 °C (data not shown).
The thermal
denaturation curves of the wild-type, nicked K86A, and intact K86A
proteins are shown in Fig. 6. Because the thermal denaturation
of the nicked protein is not reversible, only its apparent T value is compared with those of the
wild-type and intact K86A proteins (Table 2). Consistent with the
results obtained in the GdnHCl denaturation experiments, the Lys
Ala mutation did not seriously affect the thermal
stability of the protein, but the introduction of the nick dramatically
reduced it, by 16.4 °C in T
.
Figure 6:
Thermal denaturation curves of the
wild-type and K86A proteins. The apparent fraction of unfolded protein,
which was calculated as described previously (Kimura et al.,
1992), is shown as a function of temperature. , wild-type
protein;
, intact K86A protein;
, nicked K86A protein.
Thermal denaturation curves were determined at pH 3.0 by monitoring the
change in the CD value at 220 nm.
Figure 7: pH dependence of the CD spectrum of the nicked protein. The far-UV CD spectra of the nicked (a) and intact (b) K86A proteins, which were measured at 10 °C in 10 mM Gly-HCl buffer at the pH indicated in the figure, are shown.
ANS is a fluorescent dye that binds to hydrophobic regions of
proteins, and it is used as a probe for the detection of the molten
globular state of proteins. ANS effectively bound to the nicked protein
in either the D or D
` state, whereas it did not
bind to the nicked protein in the D
state (data not shown).
It bound to the nicked protein in the native state, but to a much
lesser extent. Because the CD spectrum of the nicked protein in the
D
state (Fig. 7a) is identical with that of
the nicked protein in the D
` state (Fig. 5a), the conformation of the nicked protein in
the D
state might be identical with that of the nicked
protein in the D
` state.
Figure 8: CD spectra of the N- and C-fragments. The far-UV CD spectra of the N-fragment (a) and C-fragment (b) were measured in 10 mM Gly-HCl buffer (pH 3.0), 10 mM Gly-HCl buffer (pH 3.0) containing 0.15 M NaCl, and 10 mM sodium acetate buffer (pH 5.5) at 20 °C. Because the spectra at pH 3.0 in the presence of 0.15 M NaCl were almost identical with those at pH 5.5, only the spectra at pH 3.0 in the absence of salt (brokenline) and at pH 5.5 (solidline) are shown.
The near-UV CD spectra of the N- and C-fragments at
either pH 3.0 or 5.5 were similar to that of the nicked protein in the
D state, shown in Fig. 5b (data not shown).
These results suggest that the N- and C-fragments have little tertiary
structure at pH 3.0 and pH 5.5. When the thermal denaturation of the
N-fragment was examined, either at pH 5.5 or at pH 3.0 in the presence
of 0.15 M NaCl, by monitoring the change in the CD value at
220 nm, no transition was observed in the range from 10 to 70 °C.
ANS bound to the N-fragment much more effectively than to the
C-fragment, as shown in Fig. 9. The intensity of the
fluorescence of the N-fragment at pH 3.0 was similar to that of the
nicked protein in the D
state. This result indicates that
the ANS binding site resides in the N-fragment. The intensity of the
fluorescence of the N-fragment at pH 5.5 was lower than that at pH 3.0 (Fig. 9). The formation of an additional
-helix in the
N-fragment probably affects the conformation of the ANS binding site.
In contrast, the intensity of the fluorescence of the C-fragment at pH
5.5 was slightly higher than that at pH 3.0 (Fig. 9). The
conformation of the C-fragment slightly changed at pH 5.5, so that a
weak ANS binding site was created.
Figure 9: ANS fluorescence spectra of the N- and C-fragments. The ANS fluorescence spectra of the N-fragment in 10 mM Gly-HCl buffer (pH 3.0) (thickline) and 10 mM sodium acetate buffer (pH 5.5) (thinline), and those of the C-fragment in 10 mM Gly-HCl buffer (pH 3.0) (thickbrokenline) and 10 mM sodium acetate buffer (pH 5.5) (thinbrokenline) are shown. These spectra were measured at 10 °C, as described under ``Experimental Procedures.''
To examine whether the N- and C-fragments exist in monomeric forms, they were applied to a Sephadex G-75 column at pH 3.0 and pH 5.5. The C-fragment was eluted from the column at the position at which a monomeric form of this fragment elutes, at either pH. The N-fragment was eluted from the column at the position at which a monomeric form of the N-fragment elutes, at pH 3.0. In contrast, the N-fragment was not recovered from the column at pH 5.5. It seems unlikely that the N-fragment aggregates to form a precipitate at pH 5.5, because it can associate with the C-fragment to form the nicked protein with a high yield (80%), as described below. The N-fragment may exist either in a monomeric or oligomeric form at pH 5.5. In either case, the N-fragment may have a hydrophobic surface and, therefore, may irreversibly adsorb to Sephadex G-75 at pH 5.5.
Figure 10: CD spectra of the mixtures of the N- and C-fragments. The far-UV CD spectra of the mixture of the N- and C-fragments (solidline), in which equimolar amounts of these fragments (15 µmol/liter) are mixed, are shown in comparison with the sum of the spectra of these fragments (brokenline). These spectra were measured at 20 °C in the following buffers: a, 10 mM Gly-HCl buffer (pH 3.0); b, 10 mM sodium acetate buffer (pH 5.5); c, 10 mM Gly-HCl buffer (pH 3.0) containing 0.15 M NaCl. The sum of the spectra of the N- and C-fragments was obtained from the spectra of these fragments, which were individually measured under the conditions mentioned above, as described under ``Experimental Procedures.''
Figure 11:
Elution of the reconstituted nicked
protein from the ES502C column. Elution profiles of the mixtures of the
N- and C-fragments, which were prepared either at pH 3.0 (thinline) or at pH 5.5 (thickline) in the
absence of salt, are shown. The equimolar amounts of the N- and
C-fragments (15 µmol/liter) were mixed, as described under
``Experimental Procedures.'' The concentrations of these
peptides in the resultant solution were 7.5 µM (0.075
mg/ml for the N-fragment and 0.06 mg/ml for the C-fragment), if they
were not associated with each other. An aliquot (100 µl) of this
solution was withdrawn, diluted by 10-fold with 20 mM sodium
phosphate buffer (pH 6.5), and subjected to cation-exchange HPLC using
ES502C. Elution of the reconstituted nicked protein from the ES502C
column was performed as described for the separation of the intact and
nicked proteins. The brokenline represents the
concentration of
NaSO
.
Are the conformations of these
peptides in the D state identical with those of the
corresponding regions in the intact protein in the D
state
as well? Unlike the nicked protein, the intact protein is not fully
converted to the D
state, even at an extremely low pH. Upon
acid titration, the unfolding of the intact K86A protein to the D
state begins around pH 2.5 (Fig. 7b). The
fraction of the protein in the D
state increases as the pH
decreases below 2.5. However, prior to the completion of this acid
denaturation process, the protein refolds into a molten globular state
at a pH below 1.5, because of an increase in the ionic strength by the
addition of HCl. (
)This acid-induced refolding also has been
observed for other proteins, such as apomyoglobin,
-lactamase, and
cytochrome c (Goto et al., 1990; Harada et
al., 1994). Dabora and Marqusee(1994) previously defined the
conformation of the Cys-free mutant protein of E. coli RNase
HI at pH 1.0 as an acid state (A state), which may represent a
refolding state of this protein induced by acid. Thus, it seems likely
that the CD spectrum of the intact protein at pH 1.70 (Fig. 7b) represents a mixture of the proteins in the
native and D
states. The molten globular state (A state)
may exist as well at this pH, but to a small extent. The pH dependence
of the far-UV CD spectrum of the intact protein gave the same
isodichroic point at 208 nm, with a [
] value of
-11,000, as that of the nicked protein (Fig. 7). This
result strongly suggests that the CD spectra of the intact and nicked
proteins in the D
states are identical with each other, and
therefore, the conformations of the N- and C-fragments in the D
state are identical with those of the corresponding regions in
the intact protein in the D
state.
The nicked and intact
proteins in the D state were less unfolded than that in the
U state. Generally, proteins are positively charged at around pH 2,
because most of the carboxylates are protonated at this pH. Fink et
al.(1994) have suggested that the resulting electrostatic
repulsion leads to unfolding to the D
state, but it is not
sufficient to overcome interactions, such as hydrophobic forces and
hydrogen bonds, which favor protein folding.
The addition of salt at pH 3.0, or a shift of
the pH from 3.0 to 5.5 is required for the association of the N- and
C-fragments (Fig. 10). Analyses of the conformations of these
fragments by CD (Fig. 8) suggest that the additional formation
of an -helix in the N-fragment facilitates the folding of the
C-fragment, which alone is almost fully disordered, and thereby
facilitates the association of these fragments to form the nicked
protein. The conformation of the C-fragment, either at pH 5.5 or at pH
3.0 in the presence of 0.15 M NaCl, which is designated as the
D
` state, is basically the same as that in the D
state (Fig. 8b). In contrast, the conformation of
the N-fragment either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl differs from that in the D
state in the
content of the secondary structure (Fig. 8a). This
conformation is defined as a molten globular (M) state (Kuwajima,
1989), for the following reasons. 1) It has high (native-like)
secondary structure and little tertiary structure, 2) it lacks
cooperative thermal unfolding transition, and 3) it has the ability to
bind ANS.
Schemes for the denaturation and renaturation (reconstitution) of the nicked K86A protein described above are summarized in Fig. 12.
Figure 12:
Schemes for denaturation and renaturation
(reconstitution) of the nicked K86A protein. N
C
represents the
nicked K86A protein in the native state. N
, N
, N
, and N
represent the N-fragment in the U, D
,
D
, and M states, respectively. Likewise, C
, C
, C
, and C
represent the C-fragment in the U, D
,
D
, and D
` states. The N-fragment in the M state (N
) and the C-fragment in the
D
` state (C
) are shown in parentheses, because they are stable only when they are
separated from each other. The D
` state is not shown in
these schemes, because it is identical with the D
state.
The
N-terminal segment (1-87) of the intact protein in the native
state forms three -helices (
I,
II, and
III), as
shown in Fig. 1. The contents of these helices in the N-terminal
segments are 18% for
I, 10% for
II, and 10% for
III.
Among them, the
I-helix is located at the center of the protein
molecule in the hydrophobic core region. This helix forms a
coiled-coil-like structure with the
IV-helix, and also contacts
the
-sheet. Analyses for the conformations of the small peptides
by CD suggested that the
I-helix folds independently, but the
II- and
III-helices do not. (
)In addition,
refolding studies with NMR measurements of hydrogen exchange suggested
that the
I-helix is the first to fold. (
)These results
allow us to propose that the
I-helix is the initiation site for
the folding of the intact RNase HI molecule. The
I-helix might be
at least partially formed in the N-fragment in the D
state
and the complete formation of the
I-helix in the N-fragment (M
state) may facilitate the formation of the
IV-helix in the
C-fragment and thereby facilitate the fragment association.
Alternatively, the
I-helix may be completely formed in the
N-fragment in the D
state, and may be able to induce the
formation of the
IV-helix in the C-fragment only at a pH higher
than 3.0, at which the intermolecular electrostatic repulsion between
the N- and C-fragments is suppressed.
The conformations of the
N-fragment in the D and M states may represent a folding
intermediate of the E. coli RNase HI molecule. Structural
analyses of this fragment in the D
and M states will
therefore provide valuable information on the folding process of this
protein. Because the first four
-strands and the
-helix
connecting the third and fourth
-strands (Fig. 1), which
reside in the corresponding regions of proteins with the RNase H-like
fold, are arranged identically in these protein structures, and because
all the catalytically essential carboxylates, which are
Asp
, Glu
, and Asp
in E. coli RNase HI (Kanaya et al., 1990a), are located in the
regions corresponding to the N-fragment, the folding topology
consisting of these secondary structure elements (Fig. 13) may
be evolutionarily conserved in the protein structures with an RNase
H-like fold. Katayanagi et al. (1990) have previously shown
that the crystal structure of E. coli RNase HI has a
similarity with that of DNase I (Suck and Oefner, 1986) in the folding
of a
-sheet. The similar folding topology has also been observed
in the E. coli exonuclease III structure (Mol et al.,
1995). However, these proteins may not be evolutionarily related with E. coli RNase HI, because the folding topology shown in Fig. 13is not conserved in these protein structures.
Figure 13:
A topology conserved in the RNase H-like
fold. A possible folding topology, which is evolutionarily conserved in
the protein structures with the RNase H-like fold, is shown. Arrows represent the -strands, and the cylinder represents
the
-helix.