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
Trang 1new aspects of catalytic reaction and substrate
recognition
Vladena Bauerova´-Hlinkova´1, Radovan Dvorsky´2, Dusˇan Perecˇko1, Frantisˇek Povazˇanec3
and Jozef Sˇ evcˇı´k1
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: vladena.hlinkova@savba.sk
(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 ) by x-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.
Trang 22¢,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 kcat and Km of
cleavage of the substrate analogs RpGp(S)U and
SpGp(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 kcat
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˚),
Trang 3with 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.
Crystal form I Crystal form II
Molecules in AU
Geometry statistics
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
Trang 4the 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.
Trang 5molecule 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,
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.
Trang 6molecules 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
Fig 4 (A) Electron density 2Fo–Fc(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.
Trang 7bonds 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 1 A˚ 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-conforsyn-confor-mation 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 dCp0Gp1Ap2Cp3 [37] The most important barnase subsite, labeled p2, binds the phos-phate group of the third nucleotide Occupation of the subsite for p2 gives rise to a 1000-fold increase in
kcat⁄ Km, composed of a 100-fold increase in kcatand a 10-fold decrease in Km[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,
Trang 8according 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 kcatto 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 kcatthat 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 depropro-tonated, 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.
Trang 9accepting 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
kcat 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-bindform-ing 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 kcat
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 kcatof 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
Catalytic activity at
pH 7 (%) b
T m (C)
a
From [33].bFrom [34].cFrom [56].dFrom [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.
Trang 10intermediate, 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
Refinement was performed against 95% of the data using
ran-domly excluded for the calculation of the Rfreefactor [47] The solvent molecules were modeled using warp [48] All models were checked against (2Fo–Fc; ac) and (Fo–Fc; ac) 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 Rfree 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
calculated with the Receptor Grid Generation command of