Structure of RNase Sa2 complexes with mononucleotides –
new aspects of catalytic reaction and substrate
recognition
Vladena Bauerova
´
-Hlinkova
´
1
, Radovan Dvorsky
´
2
, Dus
ˇ
an Perec
ˇ
ko
1
, Frantis
ˇ
ek Povaz
ˇ
anec
3
and Jozef S
ˇ
evc
ˇ
ı
´
k
1

cesta 21, 84551 Bratislava,
Slovakia
Fax: +421 2 59307416
Tel: +421 2 59307410
E-mail:
(Received 24 June 2008, revised 23 May
2009, accepted 29 May 2009)
doi:10.1111/j.1742-4658.2009.07125.x
Although the mechanism of RNA cleavage by RNases has been studied for
many years, there remain aspects that have not yet been fully clarified. We
have solved the crystal structures of RNase Sa2 in the apo form and in
complexes with mononucleotides. These structures provide more details
about the mechanism of RNA cleavage by RNase Sa2. In addition to
Glu56 and His86, which are the principal catalytic residues, an important
role in the first reaction step of RNA cleavage also seems to be played by
Arg67 and Arg71, which are located in the phosphate-binding site and
form hydrogen bonds with the oxygens of the phosphate group of the
mononucleotides. Their positive charge very likely causes polarization of
the bonds between the oxygens and the phosphorus atom, leading to elec-
tron deficiency on the phosphorus atom and facilitating nucleophilic attack
by O2¢ of the ribose on the phosphorus atom, leading to cyclophosphate
formation. The negatively charged Glu56 is in position to attract the pro-
ton from O2¢ of the ribose. Extended molecular docking of mononucleo-
tides, dinucleotides and trinucleotides into the active site of the enzyme
allowed us to better understand the guanosine specificity of RNase Sa2 and
to predict possible binding subsites for the downstream base and ribose of
the second and third nucleotides.
Structured digital abstract
l
MINT-7136092: RNase Sa2 (uniprotkb:Q53752) and RNase Sa2 (uniprotkb:Q53752) bind

p
Gp(S)U and
S
p
Gp(S)U by RNase T1, however, support a triester-
like mechanism that depends on the protonation of a
nonbridging phosphoryl oxygen [13].
All microbial RNases are either guanine-specific or
show a marked preference for it. Guanine binds to the
base recognition loop (residues 42–46; RNase T1 num-
bering) and forms a hydrogen bond network with the
enzyme [14]. Tyr42 (in RNase T1) or an arginine (in
RNase Sa, barnase, and binase) has an important role
in closing the guanine-binding site [10,15–17]. How-
ever, although the interactions between guanine and
the enzyme are highly specific, the molecular basis for
guanine specificity or preference is still not completely
understood [18,19].
Streptomyces aureofaciens strains BMK and R8 ⁄ 26
secrete two different guanyl-specific extracellular RNas-
es, RNase Sa and RNase Sa2 [20,21]. They hydrolyze the
phosphodiester bonds of RNA at the 3¢-side of guanosine
nucleotides in a highly specific manner. The most thor-
oughly studied is RNase Sa, which has been used as a
model for the study of protein–protein [22] and protein–
nucleotide recognition [10,23,24], protein folding and
stability [25–28], protein dynamics [29], and cytotoxicity
[30]. The mechanism of the catalytic reaction was studied
by kinetic measurements [8,9] and supported by struc-
tures of complexes of RNase Sa with guanosine 3¢-mono-

Crystal structures of RNase Sa2 with a free active site
(3D5G) and in complexes with 2¢-GMP (3DGY), exo-
2¢,3¢-GCPT (3D5I) and 3¢-GMP [crystal form I (3D4A)
was prepared by diffusion of the mononucleotide, and
crystal form II (3DH2) was obtained by cocrystalliza-
tion] were solved by molecular replacement [35] and
refined by refmac 5.0 [36] against 1.8–2.25 A
˚
data to
final R-factors between 18% and 22% (Table 1). Struc-
tures 3D5G, 3DGY, 3D5I and 3D4A have three enzyme
molecules in the asymmetric unit, and structure 3DH2
has four. RNase Sa2 consists of one a-helix (residues
14–26) and five antiparallel b-strands (residues 7–9,
54–59, 70–75, 80–83, and 91–94) (Fig. 1). The antiparallel
b-sheet, which contains three strands (residues 54–58,
71–74, and 79–83), forms the hydrophobic core of the
protein. Mononucleotides binding into the active site of
RNase Sa2 do not affect the overall fold of the protein.
Superposition of 88 corresponding CA atoms of all 16
molecules (structures 3D5G, 3DGY, 3D5I, 3D4A, and
3DH2) yielded rmsd values in the range 0.17–0.56 A
˚
.
Five N-terminal residues and loop 62–68 were removed
from the superposition, owing to high flexibility. These
segments were determined well only in molecules where
they were stabilized by a neighboring molecule.
The structure of RNase Sa2 was compared with
the structures of other microbial RNases: RNase Sa

arranged in the same way. In the complex structures,
only the active site of molecule B was accessible to the
ligand. Molecules A and C form a crystallographic
dimer by interacting through their active sites, so their
active sites are occluded (Fig. 2A). The dimer interface
is stabilized by six hydrogen bonds and a salt bridge.
In the previously solved structure of RNase Sa2 [32], a
similar dimer was formed in which Tyr87 from mole-
cule C (Tyr87C) was flipped out of its usual position
at the bottom of the active site and inserted into the
active site of molecule A. The Tyr87 aromatic ring is
positioned in the plane that is occupied by the guanine
base in the RNase Sa–mononucleotide structures. A
similar situation is also observed in 3D5G; however,
Table 1. Refinement statistics of RNase Sa2 with free active site and complexed with 2¢-GMP, 2¢,3¢-GCPT, and 3¢-GMP (crystal forms I
and II). AU, asymmetric unit; ESU, estimated standard uncertainties of atoms.
Structure 1 2 3
45
Crystal form I Crystal form II
Protein Data Bank ID 3D5G 3DGY 3D5I 3D4A 3DH2
Ligand – 2¢-GMP 2¢,3¢-GCPT 3¢-GMP 3¢-GMP
Resolution (A
˚
) 1.80 1.80 2.20 2.20 2.25
Molecules in AU
Protein 3 3 3 3 4
Mononucleotide – 1 1 1 4
Waters 505 277 167 133 125
R (%) 17.7 21.5 20.8 22 19.8
R

the superposition.
RNase
No. of corresponding
CA atoms rmsd (A
˚
)
RNase Sa2 ⁄ RNase Sa 90 0.76
RNase Sa2 ⁄ barnase 62 1.19
RNase Sa2 ⁄ binase 62 1.19
RNase Sa2 ⁄ RNase T1 47 1.80
Structures of RNase Sa2–mononucleotide complexes V. Bauerova
´
-Hlinkova
´
et al.
4158 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS
the electron density of the flipped-out Tyr87 side chain
is weaker, suggesting a lower level of occupancy. In
the structures 3DGY, 3D5I, and 3D4A, there is no
electron density for Tyr87C in this alternative confor-
mation, suggesting that the crystallographic dimer
formation is independent of Tyr87C position.
In the asymmetric unit of the RNase Sa2–3¢-GMP
crystal form II (3DH2), there are four enzyme mole-
cules (A, B, C, and D), each of which has 3¢-GMP
molecules bound in its active site. In the crystal, mole-
cules A and C, and B and D, interact through their
active sites; however, this interaction differs from that
mentioned above, as it is mediated by the 3¢-GMP
molecules present in both active sites (Fig. 2B).

the cleavage reaction. 3¢-GMP binds to the active site
of RNase Sa2 in two modes. In the first one (Fig. 3B),
seen in 3D4A and in molecules A and B of 3DH2, the
mononucleotide binds in a similar way as in RNase Sa
[10], binase [17], and barnase [37]. 3¢-GMP is in an
anti-conformation, and the ribose adopts a C2¢-endo
pucker. Guanine of 3¢-GMP forms five hydrogen
bonds: three with the amide groups of Glu40, Asn41,
and Arg42, and two with the carboxyl group of Glu43.
The base is further stabilized by interactions with the
aromatic rings of Phe39 and Tyr87, which form the
bottom of the active site. Arg42 has an important role
in guanine stabilization. In molecule B of the complex
prepared by diffusion (3D4A), the planar d-guanido
group of Arg42 undergoes a stacking interaction with
the guanine base, forming a closed conformation of
the active site [38]. The importance of this residue has
been shown by kinetic measurements of the R59A
mutation in barnase (Arg59 of barnase is structurally
equivalent to Arg42 of RNase Sa2), which abolished
85% of the wild-type barnase activity [39]. In mole-
cules A and B of 3DH2, the conformation of the
ribose is stabilized by a hydrogen bond between O4¢
and Glu56 OE1. The phosphate group of 3¢-GMP
forms several hydrogen bonds with the side chains of
Glu56, Arg67, Arg71, His86, and Tyr87. The impor-
tance of Glu56, Arg67, His86 and Tyr87 has been
investigated in RNase Sa mutants by kinetic [31] and
activity measurements (E. Heblakova, unpublished),
suggesting a similar importance for these residues in

c
(1r level), of mononucleotides exo-2¢,3¢-GCPT (3D5I) (A), 3¢-GMP (crystal form II, 3DH2) (B) and 2¢-GMP
(3DGY) (C) in the active site of RNase Sa2. For clarity, side chains of Asn41 and Arg42 are not shown. Atoms of nitrogen, oxygen and phos-
phorus are in blue, red, and cyan, respectively. In the enzyme, carbon atoms are yellow. For clarity, in the mononucleotide, carbon atoms
are green. The sulfur atom, which replaces one of the phosphate oxygens in exo-2¢,3¢-GCPT, is dark green. Hydrogen bonds between the
mononucleotide and RNase Sa2 are shown as dashed lines.
Structures of RNase Sa2–mononucleotide complexes V. Bauerova
´
-Hlinkova
´
et al.
4160 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS
molecules A and B of 3DH2). Unlike the anti-confor-
mation found in the complex with RNase Sa [24], exo-
2¢,3¢-GCPT in the active site of RNase Sa2 adopts a
syn-conformation (Fig. 3A), causing the sulfur atom to
point into the enzyme interior. The ribose O2¢ atom
of exo-2¢,3¢-GCPT forms a hydrogen bond with the
Glu56 OE1, and O3¢ forms a hydrogen bond with the
side chain of His86. The only phosphate group oxygen
forms two hydrogen bonds with Arg67 NH1 and
NH2. The sulfur is within hydrogen bonding distance
of Tyr87 OH, Arg71 NE, and His86 NE2. The side
chain of Arg34 points towards the nucleotide.
It is surprising that exo-2¢,3¢-GCPT adopts the syn-
conformation, which is proposed to be catalytic, and is
not cleaved by RNase Sa2. This is probably caused by
the presence of the sulfur atom, which points into the
active site and does not form contacts with the enzyme
equivalent to those formed by oxygen. In endo-2¢,3¢-

only in the complex with 3¢-GMP (anti-conformation).
Consequently, this substitution may account for some
of the differences observed in substrate recognition
and RNA cleavage between RNases Sa2 and Sa.
Molecular docking of nucleotides
After refinement, glucose, which had been used as a
cryoprotectant, was found in several protein molecules
in the vicinity of Tyr32, Asn33, and Arg34. The best
electron density for glucose was found in molecule C
of 3DH2 (Fig. 4A), where glucose forms two hydrogen
ABC
Fig. 4. (A) Electron density 2F
o
–F
c
(1r level) of glucose (GLC) in the vicinity of Tyr32, Asn33, and Arg34 (3DH2, molecule C). Glucose forms
two hydrogen bonds with Asn33. Dinucleotides (B) and trinucleotides (C) with highest scoring rates docked into the active site of
RNase Sa2. The trinucleotides are grouped into two clusters that differ in the position of the third nucleotide. One possible binding site is in the
area of Asp66–Gly68. The other binding site is close to the region of Tyr32, Asn33, and Arg34, which corresponds to the glucose position.
V. Bauerova
´
-Hlinkova
´
et al. Structures of RNase Sa2–mononucleotide complexes
FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS 4161
bonds with Asn33. As glucose was bound close to the
active site, we speculated that it might suggest a possi-
ble location for the substrate-binding subsite. To sup-
port this hypothesis, dinucleotides and trinucleotides
were docked into the active site.

mode was found for ribose.
Comparing guanine with adenine, cytosine and ura-
cil allowed us to better understand the guanosine spec-
ificity of RNase Sa2. In all crystal structures and
docked enzyme molecules, guanosine formed the high-
est number of hydrogen bonds of all the bases, up to
five, and had the best fit into the base-binding site. In
addition, guanine underwent a stacking interaction
with Phe39 and interacted with Arg42. Guanine forms
the most efficient hydrogen-bonding network with the
enzyme, and this seems to be very important for
proper enzyme–base binding. Other bases form a lower
number of hydrogen bonds, up to two, and have worse
fits in the RNase Sa2 active site. For the pyrimidine
bases, the base-binding site appears to be too large;
for cytosine and uracil, we observed both horizontal
shifts and rotation of the base with respect to the
plane of the guanine, by up to  40°, disrupting the
Phe39–base stacking interaction.
To find possible binding subsites of RNase Sa2, four
dinucleotides and 16 combinations of trinucleotides, all
having a guanine as the leading base, were docked into
the active site of the enzyme. In the five best-docked
dinucleotides in each protein molecule, the position of
the guanine base and most of the phosphate groups of
the first nucleotide (Gp) corresponded well with the
mononucleotides in the crystal structures. The same
was true for the ribose, which ended in a syn-confor-
mation or anti-conformation. Greater fluctuations were
observed in the positions of the ribose and base of the

Ap
2
Cp
3
[37]. The most
important barnase subsite, labeled p
2
, binds the phos-
phate group of the third nucleotide. Occupation of the
subsite for p2 gives rise to a 1000-fold increase in
k
cat
⁄ K
m
, composed of a 100-fold increase in k
cat
and a
10-fold decrease in K
m
[41]. Another important subsite
is formed by His102, which binds the base of the third
nucleotide. Comparison of the 16 RNase Sa2 docked
trinucleotides with the barnase–dCGAC complex
showed that the position of the second base of the tri-
nucleotides in RNase Sa2 is close to the corresponding
adenine in the barnase–dCGAC complex, which inter-
acts with His102. This suggests that the role of His102
in barnase is taken over by His86 in RNase Sa2
(Fig. 4C).
In RNase T1, two subsites were identified, formed

Arg67 in RNA cleavage was suggested by a site-direc-
ted mutagenesis study on RNase Sa [31]. An R65A
mutation in RNase Sa caused k
cat
to decrease by three
orders of magnitude. Because Arg65 in RNase Sa is
structurally equivalent to Arg67 in RNase Sa2, and
because, in all structures of both enzymes, these argi-
nines have almost identical conformations and are in
almost identical environments, we would expect that
an R67A substitution in RNase Sa2 would have an
effect on k
cat
that is very similar to that in RNase Sa.
In RNase Sa2–exo-2¢,3¢-GCTP, Arg67 forms a
hydrogen bond with the only oxygen in the phosphate
group of the mononucleotide, and Arg71 is within
hydrogen-bonding distance of sulfur, which replaces
the other oxygen of the phosphate group. In the other
RNase Sa2–mononucleotide structures, both arginines
form hydrogen bonds with the oxygens of the phos-
phate group of the mononucleotide (Fig. 3). At the
optimum pH of RNA cleavage by RNase Sa2, pH
7.0–7.5, both arginines are protonated, allowing them
to polarize the bonds between the oxygens of the phos-
phate group and the phosphorus atom. This leads to
an electron deficiency on the phosphorus atom,
encouraging nucleophilic attack by the electron pair of
O2¢ of the ribose (Fig. 5). The side chain of Glu56 is
turned towards O2¢ of the ribose, with OE1 within

-Hlinkova
´
et al. Structures of RNase Sa2–mononucleotide complexes
FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS 4163
accepting a proton from O2 ¢ of the ribose. NH1 and
NH2 of Arg71 form hydrogen bonds with the main
chain oxygen of Gly68, and also, in some molecules,
with the main chain oxygen of Arg67. This appears to
help to maintain the functional conformation of the
phosphate-binding site.
As originally reported by Takahashi and More [5],
in the next step, 2¢,3¢-cyclophosphate is hydrolyzed by
a water molecule that enters the active site and inter-
acts with catalytic histidine. Then, a free electron pair
of the oxygen attacks the phosphorus atom, resulting
in the opening of the cyclophosphate ring and leading
to the formation of the final product – a strand of
RNA ending with 3¢-GMP. In RNase Sa2–exo-2¢,3¢-
GCPT, there is no water molecule close to His86,
which may be attributable to the fact that exo-2¢,3¢-
GCPT is not a functional substrate. However, in
RNase Sa2–3¢-GMP (3DH2), there is a water molecule
close to His86 NE2 that forms a hydrogen bond with
O2¢ of the ribose. This water molecule, if present in
the complex with real substrate, could perform the
function of the catalytic water.
In spite of the high similarity in amino acid
sequences and tertiary structures of RNase Sa and
RNase Sa2, their kinetic and physicochemical proper-
ties differ (Table 3). To account for the differences in

tity of Arg45 (RNase Sa2) and Arg61 (binase) allows
us to consider that an R45V mutation might have a
similar effect on the k
cat
of RNase Sa2.
Summary
In this article, we have presented five structures
of RNase Sa2, one with a free active site (3D5G),
and others in complex with an analog of the reaction
Table 3. Differences in physicochemical properties of RNase Sa2
and RNase Sa.
No. of
amino
acids
Sequence
identity
(%) pI
a
Catalytic
activity at
pH 7 (%)
b
T
m
(°C)
RNase Sa2 97 53 5.3 14 41.1
c
RNase Sa 96 3.5 100 47.1
d
a

and O3¢ form hydrogen bonds with OE1 of Glu56 and
NE2 of His86, respectively. Arg67 and Arg71 interact
with the oxygens of the phosphate group, and site-
directed mutagenesis studies performed on their equiv-
alents in RNase Sa have shown that they are necessary
for the catalytic reaction. At the pH optimum for the
reaction, both arginines are protonated, facilitating
polarization of the bonds between the oxygens of the
phosphate group and phosphorus atom, leading to
electron deficiency on the phosphorus atom and,
consequently, enhancing formation of the the cyclo-
phosphate intermediate. We also propose that the
seven-fold higher efficiency of RNA cleavage by RNase
Sa than by RNase Sa2 can be at least partly explained
by the Val43 (RNase Sa) to Arg45 (RNase Sa2)
substitution. On the basis of molecular modeling
studies, we propose two possible subsites for the third
downstream nucleoside, formed by Thr61 and Arg67–
Thr69 and Tyr32, Asn33, and Arg34, respectively.
Experimental procedures
Purification, crystallization, and data collection
RNase Sa2 was purified by a procedure described by
Hebert et al. [33], with yields of 10–50 mg from 1 L of cul-
ture medium. The crystallization of RNase Sa2 with a free
active site was performed as described previously [32].
Complexes of RNase Sa2 with 2¢-GMP (3DGY), exo-2¢,3¢-
GCPT (3D5I) and crystal form I of RNase Sa2–3¢-GMP
(3D4A) were prepared by diffusion of mononucleotides into
the RNase Sa2 crystals with free active sites. The procedure
involved adding small amounts of solid mononucleotide to

factor [47].
The solvent molecules were modeled using warp [48]. All
models were checked against (2F
o
–F
c
; a
c
) and (F
o
–F
c
; a
c
)
maps and rebuilt using o [49] or xtalview [50]. Mono-
nucleotides, sulfate anions and glucose molecules were built
into clear 3r peaks in the difference electron density map
after several cycles of refinement, and their presence was
confirmed by a decrease in R and R
free
. In the final stages,
the complex structures were refined using TLS. Tempera-
ture factors, bond lengths and bond angles were restrained
according to the standard criteria employed in refmac5.
The geometry of all structures was verified with the pro-
gram procheck [51]. Analysis of the Ramachandran plot
indicated that the torsion angles for more than 90% of the
amino acids in all structures are in the most favored
regions, and that the rest lie in additionally allowed regions.

˚
in all directions
within a rectangular 40 A
˚
box, and centered at the geomet-
rical center of the bound nucleotides. Molecules of
mononucleotides 2¢-GMP, 2¢,3¢-GCPT, 3¢-GMP, 3¢-CMP,
3¢-UMP, and 3¢-AMP, dinucleotides GG, GA, GC, and
GU, and all possible combinations of trinucleotides with a
leading guanosine (16 molecules), were constructed with
module build from maestro. All ligands were prepared for
docking with the help of the ligprep module, using default
parameters with the pH set to 7.0 ± 0.5. Mononucleotides
were docked without constraints. Dinucleotides and trinu-
cleotides that bound improperly in the active site were fil-
tered out by a positional constraint of 4.5 A
˚
between the
geometrical center of the main chain nitrogen of Arg42 and
O6 of the leading guanine. Accuracy of the docking was
assessed on the basis of scoring values calculated by glide.
Analysis of the positions and conformations of docked
molecules was performed using pymol.
Acknowledgements
The authors are very grateful to Dr Jacob Bauer for
help with text editing and Dr Lubica Urbanikova for
data collection of RNase Sa2-2¢-GMP complex. This
work was supported by grant 2 ⁄ 1010⁄ 96 awarded by
the Slovak Grant Agency VEGA.
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