Tài liệu Báo cáo khoa học: Structure of RNase Sa2 complexes with mononucleotides – new aspects of catalytic reaction and substrate recognition pptx

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

1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
2 Max Planck Institute for Molecular Physiology, Dortmund, Germany
3 Faculty of Chemistry and Agricultural Technology, STU, Bratislava, Slovakia
Introduction
The microbial RNase superfamily includes more than
150 enzymes, isolated from different fungi and bacte-
ria. Most of them are small proteins that are involved
in many aspects of cellular RNA metabolism, such as
decay of mRNA, conversion of RNA precursors to
their mature form, and turnover of certain RNases [1].
The function of RNases is the hydrolysis of the 3¢,5¢-
phosphodiester bond of ssRNA. The process of RNA
cleavage has been most thoroughly investigated for
RNase T1 [2,3]. RNase T1 cleaves the O5¢-phosphodi-
ester bond after guanosine in ssRNA by a two-step
mechanism. In the first step, trans-esterification,
Keywords
binding subsite; complex structure; RNA
hydrolysis; RNase; substrate recognition
Correspondence
V. Bauerova
´
-Hlinkova
´
, Institute of Molecular
Biology, Slovak Academy of Sciences,
Du
´
bravska
´

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

(
MI:0407)byx-ray crystallography (MI:0114)
Abbreviations
2¢,3¢-GCPT, guanosine 2¢,3¢-cyclophosphorothioate; 2¢-GMP, guanosine 2¢-monophosphate; 3¢-AMP, adenosine 3¢-monophosphate; 3¢-CMP,
cytidine 3¢-monophosphate; 3¢-GMP, guanosine 3¢-monophosphate; 3¢-UMP, uridine 3¢-monophosphate; exo-2¢,3¢-GCPT, exo-guanosine
2¢,3¢-cyclophosphorothioate.
4156 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS
2¢,3¢-cyclophosphate is produced as an intermediate
product. In the second step, hydrolysis, the cyclic inter-
mediate is hydrolyzed in the presence of a water mole-
cule, yielding an RNA strand that terminates with
3¢-guanylic acid. The most important catalytic residues
in RNase T1 are Glu58 and His92, each of which
functions as an acid and a base at different steps of
the reaction [4,5]. An important role in catalysis was
also ascribed to His40. Protonated His40 interacts with
Glu58 through a hydrogen bond, enhancing the ability
of Glu58 to activate the nucleophilic attack of the
ribose O2¢ on the phosphorus atom of the phosphate
ester, leading to cyclophosphate formation. Further-
more, the positive charge of His40 helps to stabilize
the negative charge on one of the cyclophosphate
oxygen atoms [6,7]. This general acid–base mechanism
was confirmed in a number of bacterial ribonucleases
[8–12]. More recent measurements of k
cat
and K
m
of
cleavage of the substrate analogs R

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-

phosphate (3¢-GMP), guanosine 2¢-monophosphate
(2¢-GMP), and exo-guanosine 2¢,3¢-cyclophosphorothio-
ate (exo-2¢,3¢-GCPT) [10,23,24]. Glu54 and His85 were
identified as the catalytic residues acting as general
acids ⁄ base. In contrast to the situation in RNase T1,
there is no histidine analogous to His40. The importance
of Gln38, Glu54, Arg65 and His85 in RNA catalysis
has been shown by site-directed mutagenesis [31].
RNase Sa2 is homologous to RNase Sa. Their amino
acid sequence identity is 53%, and the tertiary structure
of RNase Sa2 is nearly identical to that of RNase Sa.
The amino acids involved in the catalytic reaction are
conserved in both enzymes [32]. In spite of this, the
kinetic and enzymatic properties of the two enzymes
differ [25,33,34]; for example, the catalytic constant k
cat
of RNase Sa2 at pH 7.0 is seven times lower than that of
RNase Sa [34]. To better understand the mechanism
of RNA cleavage and differences in the catalytic prop-
erties of the two RNases, we have solved the structures
of RNase Sa2 with a free active site, and in complexes
with an analog of the reaction intermediate exo-2¢,3¢-
GCPT, the catalytic cleavage product 3¢-GMP, and
2¢-GMP, which binds to the active site and functions as
an RNase Sa2 inhibitor. Extended molecular docking
of mononucleotides, dinucleotides and trinucleotides
into the active site of RNase Sa2 contributed to a better
understanding of enzyme–substrate recognition.
Results
Description of the structures

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

(2SAR), barnase (1BRN), binase (1GOY), and
RNase T1 (1RLS). As expected, the highest structural
similarity was seen with RNase Sa (rmsd of 0.71 A
˚
),
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 4157
with the largest differences (up to 3.6 A
˚
) at the
N-terminus and in region 76–78, where there is one
residue deletion in RNase Sa. Remarkably lower struc-
tural similarities were observed between RNase Sa2
and barnase (rmsd of 1.2 A
˚
), binase (rmsd of 1.2 A
˚
),
and RNase T1 (rmsd of 1.8 A
˚
) (Table 2).
Crystal packing
The asymmetric units of RNase Sa2 with a free active
site (3D5G) and in complex with 2¢-GMP (3DGY),
exo-2¢,3¢-GCPT (3D5I) and 3¢-GMP crystal form I
(3D4A) contain three enzyme molecules (A, B, and C)

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

free
(%) 24.3 24.6 26.2 26.5 26.1
ESU based on R
free
0.13 0.12 0.23 0.24 0.28
Average B (A
˚
2
) 31.6 30.6 24.3 31.8 14.7
Protein atoms 28.6 29.2 23.7 31.5 14.5
Solvent molecules 40.5 38.7 31.7 37 20.2
Geometry statistics
Bond lengths (A
˚
) 0.022 0.013 0.014 0.014 0.013
Bond angles (°) 1.892 1.471 1.555 1.520 1.610
Chiral centers (A
˚
3
) 0.112 0.087 0.114 0.108 0.106
Planar groups (A
˚
) 0.009 0.005 0.005 0.005 0.005
Fig. 1. Ribbon diagram of RNase Sa2.
Table 2. Superposition of corresponding CA atoms of RNase Sa
(2SAR, molecule A), barnase (1BRN, molecule L), binase (1GOY,
molecule A) and RNase T1 (1RLS) on RNase Sa2 (3DG4A, mole-
cule B). CA atoms that differ by more than 3 A
˚
were removed from

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).

Arg34C and Arg34D appear to play an important role
in this interaction. Their d-guanido groups form
hydrogen bonds with the phosphate group of the
3¢-GMP present in the active site of their own mole-
cule while undergoing a stacking interaction with
the guanine bases of 3¢-GMP from the neighboring
A
B
Fig. 2. Stereoview of the A ⁄ C crystallo-
graphic dimer (A, green; C, pink) in struc-
tures 3D5G, 3GDY, 3D5I, and 3D4A (A), and
in structure 3DH2 (B), in which molecules
interact through their active sites. In the
3DH2 dimer, the interaction is mediated
by the 3¢-GMP molecules present in
RNase Sa2 active sites. Residues that form
intermolecular hydrogen bonds are drawn
as sticks and labeled. Intermolecular
hydrogen bonds are shown as dashed lines.
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 4159
molecule. The A–C and B–D interfaces are further
stabilized by 10 intermolecular hydrogen bonds.
3¢-GMP in the active site of RNase Sa2
Because RNase Sa2 cleaves RNA specifically at the
3¢-side of guanosine, 3¢-GMP represents the product of

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

RNase Sa2.
In the second mode of 3¢-GMP binding, seen in mol-
ecules C and D of 3DH2, the guanine base is shifted
by 1.9 A
˚
towards Glu43 and Arg42, and the phosphate
group by about 1.4 A
˚
. However, the weaker electron
density for the mononucleotide and surrounding
residues suggests that this manner of 3¢-GMP binding
is less favorable and is probably not physiologically
relevant.
Exo-2¢,3¢-GCPT in the active site of RNase Sa2
Guanosine 2¢,3¢-cyclophosphorothioate (2¢,3¢-GCPT) is
an analog of the cyclic reaction intermediate, with one
of the two phosphate group oxygens replaced by sulfur.
There are two isomers of 2¢,3¢-GCPT, endo-2¢,3¢-GCPT
and exo-2¢,3¢-GCPT, which differ in the position of
the sulfur atom. Streptomycete RNases cleave only the
endo-isomer [24], whereas RNase T1 cleaves both the
endo-isomer and the exo-isomer, although the hydroly-
sis of the exo-isomer is much slower [40].
The guanine of exo
-2¢,3¢-GCPT is bound to the
active site in the same way as that of 3¢-GMP (3D4A,
ABC
Fig. 3. Electron density 2F
o
–F

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
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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¢-

GCPT, the positions of the sulfur and oxygen atoms
are exchanged, allowing this isomer to be cleaved. This
has also been shown by a model of endo-2¢,3¢-GCPT
built in the RNase Sa active site [24].
2¢-GMP in the active site of RNase Sa2
To obtain a set of complexes of RNase Sa2 with the
guanosine mononucleotides that were previously inves-
tigated for RNase Sa [10,23,24], we also prepared
an RNase Sa2–2¢-GMP complex. The guanine base
of 2¢-GMP is bound in the same way as in
RNase Sa2–3¢-GMP and RNase Sa2–exo-2¢,3¢-GCPT.
The nucleotide is in the syn-conformation, whereas the
ribose adopts the C3¢-endo pucker (Fig. 3C). The con-
formation of the ribose is stabilized by four hydrogen
bonds with Arg42, Arg34 and Glu56 side chains. The
phosphate group of 2¢-GMP forms a hydrogen bond
network with the side chains of Arg34, Glu56, Arg67,
Arg71, His86, and Tyr87.
The principal difference between the active sites of
RNase Sa2–3¢-GMP and RNase Sa2–2¢-GMP seems to
be in the conformation of the Arg34 side chain, which
appears to depend on whether the mononucleotide is
in the syn-conformation or anti-conformation. In
RNase Sa2–3¢-GMP (anti-conformation), the side
chain of Arg34 points outside of the active site and
does not make any contact with the mononucleotide.
In RNase Sa2–2¢-GMP (syn-conformation), the side
chain of Arg34 forms hydrogen bonds with both ribose
and phosphate. In RNase Sa, Arg34 is replaced by
Gln32, which is oriented towards the mononucleotide

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.

Seven protein molecules that form complexes with
mononucleotides in our structures were used for dock-
ing. To verify the reliability of the docking procedure,
several mononucleotides [3¢-GMP, 2¢-GMP, 2¢,3¢-
GCPT, cytidine 3¢-monophosphate (3¢-CMP), uridine
3¢-monophosphate (3¢-UMP) and adenosine 3¢-mono-
phosphate (3¢-AMP)] were docked into the free active
site of the enzyme, and the resulting models were com-
pared with those obtained from the crystal structures.
With the standard-precision setup, guanosine mono-
nucleotides were identified with the highest scores in
five of the seven enzyme molecules (Table S1). This
finding was even more pronounced when the high-
precision setup was used, in which guanosine mono-
nucleotides scored as the best in six cases. The position
of the guanine base was very similar to that found in
the crystal structures. The rmsd values of the super-
posed base atoms between the docked and observed
nucleotides were, in most cases, below 1 A
˚
. The phos-
phate groups of most docked mononucleotides were
situated in the region of the phosphate-binding site,
although the rmsd values of the phosphorus atoms
between docked and crystal nucleotides were higher,
ranging between  1A
˚
and 2.3 A
˚
. No distinct binding

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

second nucleotide. In all cases, the base of the second
nucleotide interacted with the Asp66–Thr69 loop and
with His86 (Fig. 4B).
The majority of the five best conformations of
docked trinucleotides formed two clusters (Fig. 4C). In
one cluster, the position of the ribose and the base of
the third nucleotide are located in the vicinity of Thr61
and Arg67–Thr69. In the second cluster, the ribose
and the base of the third nucleotide are close to Tyr32,
Asn33, and Arg34, which corresponds to the position
of the bound glucose. The presence of the third nucle-
otide appears to influence the position of the base of
the second nucleotide, which is turned by 90° and
sandwiched between His86 and Thr69 (Fig. 4C). The
second phosphate group of the trinucleotide is posi-
tioned between Asp66–Thr69 and Arg34 NH1 and
NH2, which are  3.2 A
˚
from the phosphate group of
the second nucleotide. This suggests that the Arg34
side chain may be important in binding the phosphate
group of the second nucleotide.
The putative binding subsites in RNase Sa2 were
compared with those found in barnase and RNase T1.
In barnase, the subsites were identified by kinetic mea-
surements [41] and confirmed by crystallization with
the tetranucleotide dCp
0
Gp
1

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

by Asn36 and Asn98. The amide group of Asn36 inter-
acts with the ribose of the leaving nucleoside, and
Asn98 is partially responsible for the cytosine prefer-
ence of the leaving nucleoside [42]. RNase Sa2 does
not have a residue equivalent to Asn98 of RNase T1.
However, Asn36 of RNase T1 correlates well with the
positions of Asn33 and Arg34 in RNase Sa2, which,
Structures of RNase Sa2–mononucleotide complexes V. Bauerova
´
-Hlinkova
´
et al.
4162 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS
according to modeling results, might form a subsite for
the third nucleotide.
Discussion
The goal of the present work was to better understand
the catalytic mechanism of RNase Sa2 and to account
for the differences in catalytic activity between RNases
Sa2 and Sa. On the basis of the crystal structures of
RNase Sa2 with mononucleotides, we can confirm that
the widely accepted reaction mechanism of guanyl-spe-
cific RNases involving glutamic acid and histidine as
important catalytic residues, as suggested by Takah-
ashi and More [5], also applies to RNase Sa2. More-
over, the structures provide more detailed information
about the role of other residues during RNA cleavage,
namely Arg67 and Arg71. Both arginines are found in
the phosphate-binding site of RNase Sa2 and are con-
served in all microbial RNases. The importance of

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

hydrogen-bonding distance of O2¢. The favorable con-
formation and distance allow Glu56 to interact with
the hydrogen atom bonded to O2¢, weakening its
attachment to the oxygen and facilitating O2¢ attack
on the phosphorus atom. In both RNase Sa2–3¢-GMP
structures (3D4A and 3DH2), His86 forms hydrogen
bonds with two oxygens of the phosphate group
(Fig. 3B), suggesting that it can be a proton donor for
the leaving O5¢ RNA strand.
Taking into consideration the conformation of both
arginine side chains in the RNase Sa2–mononucleotide
structures, Arg67 and Arg71 might also have addi-
tional roles in RNA cleavage. In three of the four
RNase Sa2–mononucleotide structures (3DGY, 3D5I,
and 3DH2), the distance between NH1 and NH2 of
Arg67 and the carboxyl group of Glu56 is below 4 A
˚
,
and the charged groups of these two residues are fac-
ing towards each other. Such a configuration might
promote a conformation of Glu56 that is favorable for
Fig. 5. The first step of RNA cleavage by RNase Sa2. At the pH optimum of RNA cleavage, 7.0–7.5, Arg67 and Arg71 are very probably pro-
tonated, Glu56 is deprotonated, and its phosphate group is negatively charged. The positively charged Arg67 and Arg71 polarize the bonds
between the oxygens of the phosphate group and phosphorus atom, causing electron deficiency on the phosphorus atom and, conse-
quently, enhancing formation of the cyclophosphate intermediate. Negatively charged Glu56 can interact with the hydrogen atom bonded to
O2¢, weakening its attachment to the oxygen and facilitating O2¢ attack on the phosphorus atom. The cyclophosphate intermediate is
formed, and the 5¢-strand of RNA is leaving from the active site. The figure was drawn with
ISIS ⁄ DRAW 2.5.
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 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

k
cat
between RNase Sa2 and RNase Sa, and to better
understand the function of the amino acids involved in
catalysis, we analyzed the active sites of RNase Sa2,
RNase Sa (2SAR, 1RSN, and 1GMP), binase (1GOY)
and barnase (1BRN) complexes. The conformations of
the residues directly involved in binding of the guanine
(residues 40–43; RNase Sa2 numbering) are almost
identical in all bacterial RNases compared (Fig. 6). In
the RNase Sa structure (2SAR), Arg40, which corre-
sponds to Arg42 of RNase Sa2, is disordered, owing
to the presence of a neighboring molecule. In the struc-
tures with different crystal packing (e.g. 1GMP),
Arg40 is ordered, forms a stacking interaction with a
guanine, and adopts a closed conformation of the
active site. Asn41 has an identical conformation in all
structures that we compared. The main role of this res-
idue is to stabilize the conformation of the loop form-
ing the base-binding site, and its importance has been
confirmed by site-directed mutagenesis studies with dif-
ferent RNases [11,43]. The main difference is found in
the position of Arg45, which is close to the base-bind-
ing site. The structural counterparts of Arg45 are
Val43 in RNase Sa and Arg61 in binase. The impor-
tance of Arg61 in binase was shown by an R61V
mutation, imitating RNase Sa, which increased the k
cat
of mutated binase seven-fold in comparison with the
wild type [18]. The structural and conformational iden-

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

From [33].
b
From [34].
c
From [56].
d
From [25].
Fig. 6. Stereoview of the active sites of
RNase Sa2 (blue, 3D4A), RNase Sa (purple,
2SAR), barnase (green, 1BRN), and binase
(brown, 1GOY). The main changes in the
active sites, which are in the Arg45 and
Arg34 positions in RNase Sa2, correspond
to Val43 and Gln32 in RNase Sa, Arg61 and
Lys26 in binase, and Ala60 and Lys27 in
barnase.
Structures of RNase Sa2–mononucleotide complexes V. Bauerova
´
-Hlinkova
´
et al.
4164 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS
intermediate, exo-2¢,3¢-GCPT (3D5I), a product of the
reaction, 3¢-GMP (3D4A and 3DH2), and the inhibitor
2¢-GMP (3DGY). In all complex structures, the guan-
ine base of the mononucleotides forms a hydrogen
bond network with the main chain nitrogens of Glu40,
Asn41, and Arg42, and OE1 or OE2 of Glu43, and the
phosphate-binding site contains Glu56, Arg67, Arg71,
His86, and Tyr87. In the exo-2¢,3¢-GCPT complex, O2¢

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

crystallization drops containing crystals of RNase Sa2 until
the concentration of the mononucleotide was close to satu-
ration. Crystal form II of RNase Sa2–3¢-GMP (3DH2) was
prepared by adding approximately twice the amount of
3¢-GMP into the crystallization drops as used for crystal
form I. This caused original crystals to dissolve; however,
new RNase Sa2–3¢-GMP crystals appeared within 1 day
[44].
Diffraction data from all crystals were collected to 1.8–
2.25 A
˚
resolution at the EMBL X31 beamline at DESY
(Hamburg), using radiation at a wavelength of 1.1 A
˚
at
100 K. The cryoprotectant solution was prepared by enrich-
ing the mother liquor to 25% glucose (w ⁄ v). The crystals
were monoclinic and belonged to the C2 space group. Opti-
mal conditions for data collection were found using the
program best [45]. denzo and scalepack were used for
processing of all datasets [46]. Data collection and process-
ing statistics for all five structures are summarized in
Table S2.
Structure determination and refinement
Structures were solved by molecular replacement using
molrep [35], with RNase Sa2 (1PY3) as a search model.
Refinement was performed against 95% of the data using
refmac5 [36]. The remaining 5% of the data were ran-
domly excluded for the calculation of the R
free

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.

The final refinement statistics for all five structures are
given in Table 1. To evaluate the similarity of the struc-
tures, CA atoms of all molecules were superposed with
molecule A from 3D5G with the program multiprot [52].
Five N-terminal CA atoms and seven CA atoms in loop
61–67 were excluded from superposition because they were
not modeled in most of the molecules, owing to poor elec-
tron density. All figures were drawn by pymol [53]. The
numbering of amino acids is according to RNase Sa2 unless
indicated otherwise.
Molecular docking of the nucleotides into the
active site of RNase Sa2
Module glide [54] from maestro [55] was used for mole-
cular docking. Structures of RNase Sa2 in which the ligand
was found in the active site (B molecules of the complex
structures 3DGY, 3D5I, and 3D4A, and all four molecules
of 3DH2) were selected for docking. All nonprotein mole-
cules (nucleotides, sulfates, glucoses, and waters) were
removed, and input files containing protein molecules were
preprocessed using the Protein Preparation command of
glide. Interactions of probe atoms with proteins were
calculated with the Receptor Grid Generation command of
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 4165
glide at the points of a regular three-dimensional grid
around the active site, spaced by 1 A

˚
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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Supporting information
The following supplementary material is available:
Table S1. Molecular docking of mononucleotides
3¢-GMP, 2 ¢-GMP, exo-2¢,3¢-GCPT, 3¢-CMP, 3¢-UMP
and 3¢-AMP into the active site of RNase Sa2, using
standard-precision and high-precision setups of module
glide [54] from maestro [55].
Table S2. Data processing statistics of RNase Sa2–
mononucleotide complex structures.
This supplementary material can be found in the

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Structures of RNase Sa2–mononucleotide complexes V. Bauerova
´
-Hlinkova
´
et al.
4168 FEBS Journal 276 (2009) 4156–4168 ª 2009 The Authors Journal compilation ª 2009 FEBS