Treatment of unresectable cancer by traditional chemotherapy employs small molecules that interfere with DNA transcription; this type of treatment, Keywords crystal structure; proteolysi
Trang 1interaction with the cytosolic ribonuclease inhibitor
Ulrich Arnold, Franziska Leich*, Piotr Neumann, Hauke Lilie and Renate Ulbrich-Hofmann
Department of Biochemistry and Biotechnology, Martin-Luther University Halle-Wittenberg, Halle, Germany
Introduction
About 10 million new cases of cancer are diagnosed
worldwide annually, and cancer is the second most
frequent cause of death after cardiovascular diseases
Treatment of unresectable cancer by traditional chemotherapy employs small molecules that interfere with DNA transcription; this type of treatment,
Keywords
crystal structure; proteolysis; ribonuclease
inhibitor; stoichiometry; RNase A;
tandem enzyme
Correspondence
U Arnold, Department of Biochemistry and
Biotechnology, Martin-Luther University
Halle-Wittenberg, Kurt-Mothes Str 3, 06120
Halle, Germany
Fax: +49 345 5527303
Tel: +49 345 5524865
E-mail: ulrich.arnold@biochemtech.
uni-halle.de
Website: http://www.biochemtech.
uni-halle.de/biotech
Present addresses
*Institute of Medical Immunology,
Martin-Luther-University Halle-Wittenberg,
Magde-burger Str 2, 06097 Halle, Germany
Institute of Microbiology and Genetics,
Georg-August University Go¨ttingen,
Justus-von-Liebig-Weg 11, 37077
Go¨ttingen, Germany
Database
Structural data are available in the Protein
Data Bank under the accession numbers
3MX8, 3MWR, and 3MWQ
(Received 27 August 2010, revised 3
November 2010, accepted 8 November
2010)
doi:10.1111/j.1742-4658.2010.07957.x
Because of their ability to degrade RNA, RNases are potent cytotoxins The cytotoxic activity of most members of the RNase A superfamily, how-ever, is abolished by the cytosolic ribonuclease inhibitor (RI) RNase A tan-dem enzymes, in which two RNase A molecules are artificially connected by
a peptide linker, and thus have a pseudodimeric structure, exhibit remark-able cytotoxic activity In vitro, however, these enzymes are still inhibited by
RI Here, we present the crystal structures of three tandem enzymes with the linker sequences GPPG, SGSGSG, and SGRSGRSG, which allowed us
to analyze the mode of binding of RI to the RNase A tandem enzymes Modeling studies with the crystal structures of the RI–RNase A complex and the SGRSGRSG-RNase A tandem enzyme as templates suggested a
1 : 1 binding stoichiometry for the RI–RNase A tandem enzyme complex, with binding of the RI molecule to the N-terminal RNase A entity These results were experimentally verified by analytical ultracentrifugation, quanti-tative electrophoresis, and proteolysis studies with trypsin As other dimeric RNases, which are comparably cytotoxic, either evade RI binding or poten-tially even bind two RI molecules, inactivation by RI cannot be the crucial limitation to the cytotoxicity of dimeric RNases
Abbreviations
BS-RNase, bovine seminal RNase; ds-RNase A, domain-swapped RNase A; FAM-AUAA-TAMRA, 6-carboxyfluorescein-dArU(dA) 2
-6-carboxytetramethylrhodamine; RATE, RNase A tandem enzyme; RI, ribonuclease inhibitor.
Trang 2however, is often accompanied by severe side effects
[1] Antibody-based therapeutics, which target a variety
of proteins (mostly on the cell surface), are much more
selective, thereby reducing the systemic toxicity of the
compounds In the search for new cytotoxic
therapeu-tics, RNases are considered to be powerful –
nonmuta-genic – compounds by virtue of their RNA-digesting
activity [1,2] Whereas cell death was expected to be
caused by ‘simple’ inhibition of the translation of the
genetic information into proteins by unspecific RNA
degradation, RNases were found to induce
caspase-mediated apoptosis [3,4], probably by targeting
non-coding RNAs [5] Interestingly, members of the
RNa-se A superfamily, which are basic proteins, show a
specificity in their cytotoxic action for malignant cells
[4] However, it is still unclear whether the unusual
intracellular trafficking of the endocytosed RNases in
transformed cells [6] or the altered cell surface
carbo-hydrate and lipid composition [7], which results in an
increase in negative charge, and thus favors the
bind-ing of the RNases [2], is responsible for the specific
action Unfortunately, the cytotoxicity of these RNases
is limited by several factors at the cellular level [8],
including restricted internalization into the cell, release
from the endosomes, and inhibition by the cytosolic
ribonuclease inhibitor (RI) RI is an abundant 50-kDa
protein that binds the mammalian members of the
RNase A superfamily extraordinarily tightly, with KD
values in or below the picomolar range [9–12]
Conse-quently, RI evasion is considered to be crucial for
cytotoxic efficacy [13] In fact, Onconase (Tamir
Bio-technology, Inc., Monmouth Junction, NJ, USA), an
RNase A homolog from the Northern leopard frog,
and the only naturally occurring dimeric RNase,
bovine seminal RNase (BS-RNase), evade RI binding
and are cytotoxic, as are genetically engineered
RNase A variants with decreased affinity for RI
[13–15] Among the numerous approaches that were
conceived to improve the cytotoxicity of mammalian
RNases, the generation of pseudodimeric RNase A
tandem enzymes (RATEs) proved to be very efficient
[16] In contrast to the noncytotoxic monomeric
RNa-se A, RATEs show remarkable cytotoxicity (IC50
val-ues ‡ 13 lm for K-562 cells [16]), as do the dimeric
BS-RNase (IC50 1 lm for malignant SVT2
fibro-blasts [14]) and artificially domain-swapped RNase A
(ds-RNase A) dimers (IC50 0.5 lm for HL-60 cells
[17]) Like these, RATEs consist of two RNase entities
However, by means of gene duplication, RATEs
consist of a single polypeptide chain [16] In this way,
dissociation of the RNase dimers is prevented The
dimeric structure was shown to be essential for a
cyto-toxic effect of BS-RNase [18], and dimerization by
domain swapping has been suggested to cause cytotox-icity by improved cellular uptake [19] Tandemization considerably enhances endocytotic internalization into the cells [20], and potentially impedes binding by RI Despite their clear cytotoxic action and their dimeric structure, however, RATEs were inhibited by RI
in vitro at concentrations comparable to those that inactivate monomeric RNase A [16] Whereas (at least C-terminally swapped) ds-RNase A dimers are sug-gested to bind two RI molecules in vitro, resulting in
an inactive complex [19], BS-RNase evades RI binding [14,21]
To elucidate the mode of binding of RI and RATEs, the crystal structures of various RATEs differing in linker sequence length and amino acid composition (GPPG, SGSGSG, or SGRSGRSG) were solved On the basis of these structures, modeling studies consider-ing the bindconsider-ing of one or two RI molecules to SGRSGRSG-RATE were performed, and were com-plemented by analytical ultracentrifugation, 2D elec-trophoresis, and proteolysis experiments The studies revealed a 1 : 1 stoichiometry, with binding of the RI molecule to the N-terminal RNase A entity
Results
Crystal structure of RATEs RATEs are composed of two RNase A molecules that are covalently linked by a peptide linker, resulting in
a single polypeptide chain [16] Three RATEs with linker sequences that differ in charge or flexibility (GPPG, SGSGSG, and SGRSGRSG) were selected for determination of the crystal structures These RATEs were previously shown to be active [48–69%, relative to monomeric RNase A, with RNA as sub-strate; 1–5%, relative to monomeric RNase A, with 6-carboxyfluorescein-dArU(dA)2 -6-carboxytetramethyl-rhodamine (FAM-AUAA-TAMRA) as substrate] and cytotoxic (IC50 values between 12.9 lm and 40.0 lm with mammalian K-562 cells) All three variants showed thermodynamic stabilities similar to that of monomeric RNase A, and were inactivated by RI (Ki£ 2.5 nm, as shown for SGRSGRSG-RATE) [16] GPPG-RATE, SGSGSG-RATE and SGRSGRSG-RATE were successfully crystallized as described in Experimental procedures, all yielding the same crystal form The crystals were highly isomorphic, and the structures could be solved at 2.10, 1.85 and 1.68 A˚ resolution, respectively (Table S1) The structure of SGRSGRSG-RATE, which had been obtained at the highest resolution, was used as a model to refine the structures of the RATEs with the GPPG or SGSGSG
Trang 3linker by the Fourier difference method Comparison
of the structures (Fig S1) revealed no significant
dif-ferences, with the exception of the linker region As
expected, the SGRSGRSG and GPPG linkers form
loops between the two RNase A entities In contrast,
the SGSGSG linker could not be completely defined,
indicating its particularly high flexibility Interestingly,
all RATEs showed the same alignment of the two
RNase A entities Consequently, the lengths and
com-positions of the linker sequences used have no impact
on their arrangement For this reason, the following
studies focused on SGRSGRSG-RATE, the structure
of which was determined with the highest resolution
(Fig 1A; Table S1)
Surprisingly, the orientation of the individual
RNase A entities within the asymmetric unit, which
were positioned almost perpendicular to each other,
was found to be the same as in both the RNase A and
RNase B (a glycosylated form of the enzyme) crystal
structures [22,23]
The contact surface between the two RNase A
enti-ties within one RATE molecule consists of a-helix III
(residues 51–57), a b-strand (residues 61–63) and a
loop region (residues 75–79) of the N-terminal entity,
and a-helix I¢ (residues 4¢–12¢), a loop region
(resi-dues 13¢–18¢) and the end of a-helix II¢ (resi(resi-dues
29¢–32¢) of the C-terminal entity (see Fig 1B for
num-bering of the amino acids in the RATEs) Interestingly,
the crystal structure provides no clear indication of the
reason for the decrease in catalytic activity as
com-pared with RNase A Neither of the two active sites is
blocked by the interactions, and even though His12¢,
which is an essential component of the active site [24],
is part of helix I¢, its side chain points away from the interface Nevertheless, the tandemization is undoubtedly the reason for the decreased activity, as concluded from the considerable increase in activity after liberation of the individual RNase A entities by trypsin (Table 1)
Modeling of the RI–RATE complex
As intensive attempts at the crystallization of the com-plex mixture of RI and RATEs have failed so far, models for RI–RATE complexes were derived from the crystal structure of SGRSGRSG-RATE (Fig 1) and the porcine RI–RNase A complex [25] by superim-posing the RNase A structures with the program lsqman [26]
A
B
124 ′
1 ′ 124
1
Linker
180°
N-terminal RNase A entity C-terminal RNase A entity
Fig 1 Crystal structure and amino acid numbering of SGRSGRSG-RATE The crystal structure of SGRSGRSG-RATE (A) was produced with
PYMOL [42] The N-terminal RNase A entity is shown in ruby, the C-terminal RNase A entity is shown in orange, and the linker is shown in blue (B) The amino acids within the RATEs are numbered 1–124 for the N-terminal RNase A entity and 1¢–124¢ for the C-terminal RNase A entity; the amino acids of the linker, which differs in length between the various RATEs, are not numbered.
Table 1 Catalytic efficiency of SGRSGRSG-RATE upon cleavage of the SGRSGRSG linker by trypsin in the absence or presence of RI The activity assay was carried out as described in Experimental procedures.
Sample
kcat⁄ K M ( M )1Æs)1) Before trypsin treatment
After trypsin treatment RNase A (2.5 ± 0.5) · 10 7 Not determined SGRSGRSG-RATE (4.8 ± 0.8) · 10 6 (2.1 ± 0.6) · 10 7 SGRSGRSG-RATE
+ RI (1 : 1)
(9.7 ± 1.5) · 10 5 (1.8 ± 0.4) · 10 7 SGRSGRSG-RATE
+ RI (1 : 250)
(1.9 ± 0.6) · 10 5
(2.9 ± 0.5) · 10 5
Trang 4As shown in Fig 2A, binding of an RI molecule to
the N-terminal RNase A entity of SGRSGRSG-RATE
is possible without restrictions In contrast,
superimpo-sition of the structure of the RI–RNase A complex
and the C-terminal RNase A entity of
SGRSGRSG-RATE indicates considerable steric hindrance
(Fig 2B) In accordance with this result, modeling of
the complex between SGRSGRSG-RATE and two RI
molecules (Fig 2C) revealed severe steric clashes The
hydration shell, which was not considered in these
modeling studies, may enhance the mismatch effect
even further Consequently, a 1 : 1 complex with
bind-ing of the RI molecule to the N-terminal RNase A
entity of the RATE seems most likely
In solution, however, the latitude of the RNase A
entities may be different As the molecules of
mono-meric RNase A, which are highly ordered in the crystal
[22] and are arranged like the RNase A entities in the
RATEs, completely dissociate in solution, it seems
likely that the close contact of the RNase A entities in
the RATEs is also lost in solution This assumption is
supported by shape complementarity (sc) calculations
for the two interacting molecular surfaces, performed
with the program sc from the ccp4 suite [27] The
obtained sc value of 0.400 indicates weak binding of
the two entities of the RATE Therefore, the molecule
might become more relaxed in solution, thereby
enabling RI binding to both the N-terminal and the
C-terminal RNase A entities The distance between the
C-terminus of the first and the N-terminus of the
second RNase A entity was estimated to be 11.6 A˚
in the crystal structures of all RATEs (Fig S1), but it
might increase up to 30 A˚ (SGRSGRSG linker) when
the peptide is fully stretched In the closest modeled
arrangement of two RI–RNase A complexes that is
possible without steric clashes, the distance between
the C-terminus of the RNase A in the first complex
and the N-terminus of the RNase A in the second
complex is about 6 A˚, i.e less than the distance that would be covered by two (stretched) amino acids
As the RATEs that have been used so far [16,20] contain linker sequences of four (GPPG) to eight (SGRSGRSG) amino acids, a more relaxed conforma-tion of the RATEs in soluconforma-tion and, consequently, a stoichiometry for RI–RATE higher than 1 : 1, cannot
be excluded by the modeling studies However, the linker keeps the two RNase A entities close to each other not only in the crystal but also in solution, thereby supporting their interactions As the intra-cellular concentration of macromolecules is close to the crystallization conditions [28], intensive interactions between the two RNase A entities are also likely in solution
Analytical ultracentrifugation Analytical ultracentrifugation was used to determine the stoichiometry within the RI–SGRSGRSG-RATE complex experimentally Experiments with 1.5 lm RI and different concentrations of RATE (0–3 lm) yielded a maximum sedimentation velocity (sapp) of 5.22 S at 1.5 lm SRGSGRSG-RATE (Fig 3), i.e at a molar ratio of 1 : 1 of RI and SGRSGRSG-RATE in the complex
Analysis of the RI-binding stoichiometry by gel electrophoresis
For verification of the 1 : 1 stoichiometry in the RI– RATE complex, RNase A and SGRSGRSG-RATE were incubated in the presence of RI and separated by native PAGE (Fig 4) Whereas a two-fold molar quantity of RI (Fig 4, lanes 1 and 3) leaves unbound
RI in both cases, no free RI was detectable at an equi-molar ratio (Fig 4, lanes 2 and 4), confirming the 1 : 1 binding stoichiometry in the RI–RATE complex
Fig 2 Alignment of SGRSGRSG-RATE with the RI–RNase A complex The crystal structure of the porcine RI–RNase A complex (Protein Data Bank entry: 1DFJ) was aligned with the N-terminal (A) or the C-terminal (B) RNase A entity of the tandem enzyme or with both
RNa-se A entities (C) The RI molecule, which binds to the N-terminal RNaRNa-se A entity, is shown in bright green, and the RI molecule, which binds
to the C-terminal RNase A entity, is shown in pale green The N-terminal and C-terminal RNase A entities are shown in ruby and orange, respectively, and the linker is shown in blue.
Trang 5Additionally, the bands containing the RI–RNase A
or RI–SGRSGRSG-RATE complexes (Fig 4, lanes 2
and 4) were excised and subjected to SDS⁄ PAGE, in
which the noncovalent complexes dissociate (Fig S2)
The ratios of band intensities of RI–RNase A and
RI–SGRSGRSG-RATE were (2.9 ± 0.5) : 1 and
(1.3 ± 0.2) : 1, respectively These values are slightly
lower than calculated for a 1 : 1 stoichiometry from
the molecular masses (3.6 and 1.7), because of the
indi-vidual staining properties of RNase A,
SGRSGRSG-RATE, and RI, but clearly indicate a 1 : 1 binding
stoichiometry in the RI–RATE complex
Assessment of the functionality of the RNase A entities of SGRSGRSG-RATE by tryptic cleavage
of the linker RNase A resists proteolytic attack by trypsin at 25C [29], whereas the flexible linker in SGRSGRSG-RATE
is expected to be cleaved at the two potential cleavage sites (C-terminal to the two Arg residues) This specific cleavage should allow liberation of the RNase A enti-ties and thus allow evaluation of their activity loss by
RI binding in the complex In fact, cleavage by trypsin occurred exclusively within the linker sequence (Fig 5), and the cleavage products were identified as RNase A-SGR and SG-RNase A by MS (13 986 and
13 829 Da in comparison with the theoretical values of
13 983 and 13 826 Da); that is, trypsin cleaves the lin-ker C-terminally to both Arg residues Interestingly, the kcat⁄ KM value of SGRSGRSG-RATE increased upon cleavage of the linker by trypsin to about that of RNase A (Table 1) From this activation, it can be unambiguously concluded that the RNase A entities within the RATEs are catalytically active and that the activity decrease [16] is a result of the tandemization
Accessibility of the linker of SGRSGRSG-RATE in the presence of RI
The accessibility of the linker sequence in SGRSGRSG-RATE was evaluated in the presence of
RI, first by SDS⁄ PAGE (Fig 5) As observed for the RNase A monomers, RI also resisted proteolytic attack by trypsin (not shown), whereas the linker sequence of SGRSGRSG-RATE was cleaved and
Fig 3 Analysis of the stoichiometry of the RI–SGRSGRSG-RATE
complex by analytical ultracentrifugation Formation of the complex
between SGRSGRSG-RATE and RI (1.5 l M ) was analyzed in the
absence and in the presence of different amounts of the tandem
enzyme (0–3 l M ) in 0.1 M sodium phosphate buffer (pH 6.5)
con-taining 2 m M dithiothreitol and 0.5 m M EDTA The sedimentation
velocity was analyzed at 40 000 r.p.m (130 000 g) and 20 C.
RI - RATE complex
RI - RNase A complex RI
Fig 4 Analysis of the stoichiometry of the RI–SGRSGRSG-RATE
complex by native PAGE Lanes 1 and 2: complex of RNase A
(100 pmol) with RI (200 and 100 pmol, respectively) Lanes 3 and
4: complex of SGRSGRSG-RATE (‘‘RATE’’, 100 pmol) with RI (200
and 100 pmol, respectively) Neither unbound RNase A nor
SGRSGRSG-RATE is visible in the native PAGE gel [16].
RI RATE
Fragments
1
M 2 Fig 5 Analysis by SDS ⁄ PAGE of the impact of RI on the tryptic cleavage of SGRSGRSG-RATE SGRSGRSG-RATE (‘‘RATE’’) was incubated with trypsin in the absence (lane 1) or in the presence (lane 2) of RI, as described in Experimental procedures Lane M shows the molecular mass marker proteins lactalbumin (14.4 kDa), soybean trypsin inhibitor (21 kDa), carbonic anhydrase (30 kDa), ovalbumin (43 kDa), BSA (66 kDa), and phosphorylase b (97 kDa) The resulting cleavage products (see text) were combined and denoted as ‘fragments’.
Trang 6yielded the same fragment pattern as in the absence of
RI (Fig 5)
Next, the catalytic activity of the
RI–SGRSGRSG-RATE complex was studied before and after treatment
with trypsin (Table 1) Under the conditions applied,
the activity of SGRSGRSG-RATE was decreased by
80% and 96% in the presence of RI at ratios of 1 : 1
and 1 : 250, respectively After tryptic cleavage of the
linker, considerable activity was regained at an
equi-molar RI⁄ SGRSGRSG-RATE ratio, indicating
the release of an active RNase A entity from the
RI–SGRSGRSG-RATE complex by trypsin In
con-trast, no significant increase in activity could be
obtained at a 250-fold excess of RI, which proves that
the released RNase A entity is, like the natural
RNa-se A, RNa-sensitive to RI binding
Finally, the mode of binding of RI to
SGRSGRSG-RATE was studied by cation exchange
chromatogra-phy Under the conditions applied, RNase A,
SGRSGRSG-RATE and their complexes with RI elute
at distinct, separated elution times (Fig S3A) After
tryptic treatment of the RI–SGRSGRSG-RATE
com-plex, a new peak (peak 1 in Fig S3A) emerged at an
elution time similar to that of RNase A Analysis by
MS yielded a mass of 13 830 Da (Fig S3B), which
unambiguously identified the released RNase A entity
as SG-RNase A (13 826 Da), i.e the C-terminal
RNase A entity, and thus proved binding of RI to the
N-terminal RNase A entity of SGRSGRSG-RATE
Discussion
The covalent linkage of two RNase A molecules, which
are not cytotoxic in the monomeric form, by a peptide
linker has been proven to endow cytotoxicity [16]
simi-lar to that of the natural dimeric BS-RNase, in which
the RNase entities are linked by two intermolecular
disulfide bonds [14], or ds-RNase A dimers, in which
the RNase A entities are noncovalently held together
by swapping of the N-terminal or C-terminal ‘domains’
[19] BS-RNase evades RI binding in vitro, which is
regarded as a reason for its in vivo cytotoxicity Upon
reduction of the intermolecular disulfide bonds,
however, the monomers slowly dissociate and become
susceptible to inhibition by RI [21], losing their
cyto-toxic properties [18] In contrast to BS-RNase,
ds-RNase A dimers show an affinity for RI comparable
to that of RNase A, and the formed RI–ds-RNase A
dimer complex apparently possesses a 2 : 1
stoichiome-try [19] Despite slow dissociation of the ds-RNase A
dimers, they proved to be cytotoxic as well [17],
because of improved interaction of the dimers with the
negatively charged cell membrane (dimerization
increases the local concentration of RNase molecules
on the cell surface), thereby favoring their endocytosis [19] The stoichiometry of RI binding to RATEs and the role of the RI–RATE complex in the in vitro inacti-vation of RATEs have so far been obscure
The crystal structures of three different RATEs (Figs 1 and S1) revealed that the linkers do not con-strain the ability of the RNase A entities to adopt the same orientation in the crystal as monomeric RNase A
On the other hand, the decreased activity of the RATEs
as compared with RNase A ([16] and Table 1) indicate
a negative influence of the tandemization on the cata-lytic efficiency The recovery of activity after proteocata-lytic cleavage of the linker clearly proves that the decreased activity is a result of tandemization Interestingly, the activities of both BS-RNase [30] and ds-RNase A dimers [19] are also decreased by about 60% and 70%, respectively, in comparison with RNase A
Modeling studies on the RI–RATE interactions on the basis of the crystal structure of SGRSGRSG-RATE (Fig 2) suggest a 1 : 1 binding stoichiometry in the RI–RATE complex, with binding of the RI mole-cule to the N-terminal RNase A entity These conclu-sions were unambiguously confirmed by experiment The results of ultracentrifugation (Fig 3) and electro-phoresis analyses (Fig 4) clearly show that RATEs are able to bind one RI molecule only, corresponding to a
1 : 1 binding stoichiometry in the RI–RATE complex (at least at protein concentrations £ 6.67 lm; that is, the KD value for the C-terminal RNase A entity
is ‡ 6.67 lm, whereas KD values of mammalian RI–RNase complexes are in the picomolar range or below [9,12]) By tryptic cleavage of the linker in the RI–SGRSGRSG-RATE complex and MS analysis of the released RNase A entity (Figs S3A,B), the sug-gested binding position of RI at the N-terminal
RNa-se A entity was verified The experimentally determined binding stoichiometry corroborates the ori-ginal idea in the design of RATEs [16] As the intracel-lular concentration of RI is about 4 lm [31], sufficient activity may remain in vivo to explain the cytotoxicity
of the tandem constructs, even though activity mea-surements in the presence of RI indicated a decrease in activity that was larger than expected (Table 1) More-over, tandemization has been shown to dramatically improve endocytosis efficiency [20]
In summary, the three types of dimeric RNase, which are comparably cytotoxic, differ fundamentally
in their RI binding: BS-RNase binds no RI, ds-RNase A dimers supposedly bind two RI molecules, and RATEs bind one RI molecule Therefore, RI evasion cannot be the pivotal determinant for cytotox-icity of dimeric RNase variants Rather, improved
Trang 7endocytosis in comparison with monomeric RNase A
seems to be the decisive factor Dimerization by a
non-reducible covalent linkage, which prevents dissociation,
renders RATEs superior to other types of RNase
dimers as cytotoxic agents
Experimental procedures
Proteins and chemicals
RNase A from Sigma (Taufkirchen, Germany) was purified
on a SOURCE S FPLC system (Amersham Biosciences,
Uppsala, Sweden) Growth media were from Difco
Labora-tories (Detroit, MI, USA) Escherichia coli strain
BL21(DE3) was from Stratagene (Heidelberg, Germany)
FAM-AUAA-TAMRA was from Metabion International
AG (Martinsried, Germany) All other chemicals were of
the purest grade commercially available
Expression, renaturation and purification of
RATEs
The experimental procedures for expression and
renatur-ation of RATEs have been described previously [16] The
proteins were purified on a SOURCE S FPLC system
(50 mm Tris⁄ HCl, pH 7.5, with a linear gradient of
0–500 mm NaCl)
Expression and purification of RI
The plasmid pET–22b(+), which contains the gene for RI,
was a gift from R T Raines (UW-Madison, WI, USA) RI
was purified by procedures similar to those described
previ-ously [12] Briefly, the plasmid was transformed into E coli
BL21(DE3) cells, and a single colony was used to inoculate
LB medium (25 mL) containing ampicillin (200 lgÆmL)1)
An overnight culture, grown at 37C and 180 r.p.m for
12 h, was used to inoculate cultures of TB medium (1 L)
containing ampicillin (200 lgÆmL)1) These cultures were
grown at 37C and 180 r.p.m up to D600 nm‡ 3.0
Expres-sion of the ri gene was induced by addition of isopropyl
thio-b-d-galactoside to a final concentration of 0.5 mm, and
the cultures were grown at 15C and 120 r.p.m for 24 h
Bacteria were harvested by centrifugation (7000 g for
15 min), and resuspended in 200 mL of 20 mm Tris⁄ HCl
buffer (pH 7.8) containing 10 mm EDTA, 10 mm
dith-iothreitol, 100 mm NaCl, and 0.04 mm
phenylmethanesulfo-nyl fluoride After lysis of the bacteria by three passages
through a Gaulin Lab 40 (APV, Lu¨beck, Germany), cell
debris was removed by centrifugation (48 000 g, 20 min,
4C) The supernatant after the centrifugation step, which
contained the soluble RI, was loaded onto an RNase A
affinity column For affinity chromatography, RNase
A was attached covalently to the resin of a 5-mL
HiTrap NHS-ester column (Amersham Biosciences), follow-ing the manufacturer’s protocol RI was eluted in 100 mm sodium acetate buffer (pH 5.0) containing 3 m NaCl,
10 mm dithiothreitol, and 1 mm EDTA, after extensive washing with 50 mm KH2PO4 buffer (pH 6.4) containing
1 m NaCl, 10 mm dithiotheitol, and 1 mm EDTA The pro-tein eluted from the RNase A affinity resin was dialyzed for 16 h against 10 L of 20 mm Tris⁄ HCl buffer (pH 7.5) containing 10 mm dithiothreitol and 1 mm EDTA, and purified further by anion exchange chromatography with a Mono Q column (Amersham Biosciences; 20 mm Tris⁄ HCl,
pH 7.5, containing 10 mm dithiothreitol and 1 mm EDTA, with a linear gradient of 0–1 m NaCl) The purity of the eluted RI was determined to be > 99% by SDS⁄ PAGE (data not shown)
Crystallization
Crystals of RATEs were obtained by hanging-drop vapor diffusion over 30% (w⁄ v) poly(ethylene glycol)-8000 con-taining 200 mm (NH4)2SO4 The hanging-drop solution contained a mixture of purified RATE (2 lL; 10 mgÆmL)1
in 10 mm Tris⁄ HCl, pH 7.0) and crystallization solution (2 lL) Diffraction-quality crystals grew within 6 days at
13C to a size of 0.1 · 0.1 · 0.1 mm
Structure determination
A redundant dataset of a RATE crystal was collected at
100 K on a flash-frozen crystal by transferring the crystal rapidly into a cryoprotectant containing mother liquor made up to 20% (v⁄ v) glycerol The crystals diffracted up
to 1.68 A˚ resolution with Cu Ka radiation (k = 1.5418 A˚), with a rotating-anode source (RA Micro 007; RigakuMSC, Sevenoaks, Kent, UK) and image plate detector (R-AXIS IV++; RigakuMSC) Oscillation photographs were integrated, merged and scaled with mosflm and scala, respectively (details are given in Table S1), from the ccp4 suite [32]
The crystals of RATEs crystallize in space group C2 (Table S1) The structure was determined by the molecular replacement method with data between 20 and 2.5 A˚, using the RNase A structure (Protein Data Bank code: 1SRN [33])
as the search model, with phaser [34] The molecular replace-ment search solution showed two RNase molecules (corre-sponding to one tandem enzyme molecule) occupying the asymmetric unit (Matthews coefficient of 1.86 A˚3⁄ Da, corre-sponding to 33.3% solvent content) The structure was man-ually rebuilt and verified against a simulated annealing omit map as well as SIGMAA-weighted [35] difference Fourier maps, with the o and coot programs [36,37] The final refine-ment was performed with refmac from the ccp4 suite [32], with TLS parameterization Both cns and refmac used the same Rfreeset (randomly chosen 5% of the reflections) Six
Trang 8residues displaying dual conformations were modeled The
stereochemistry of the structure was assessed with procheck
[38] (Ramachandran plot statistics:88% of all amino acids
in favored regions, and12% in allowed regions)
Analytical ultracentrifugation
For analysis of the stoichiometry of the
RI–SGRSGRSG-RATE complex, RI (1.5 lm) and SGRSGRSG-RI–SGRSGRSG-RATE
(0–3 lm) were incubated in 0.1 m sodium phosphate buffer
(pH 6.5) containing 2 mm dithiothreitol and 0.5 mm
EDTA Formation of the complex between
SGRSGRSG-RATE and RI was studied with a Beckman Optima XL-A
analytical ultracentrifuge (Palo Alto, CA, USA), using an
An-50Ti rotor The experiment was carried out at
40 000 r.p.m (130 000 r.p.m.) and 20C for calculation of
the sedimentation velocity (absorption at 280 nm) Data
were analyzed with the software provided by Beckman
Instruments (Palo Alto, CA, USA)
Analysis of the RI-binding stoichiometry by gel
electrophoresis
To analyze the stoichiometry of the complex between RI
and RATEs, 100 or 200 pmol of RI was incubated either
with 100 pmol of SGRSGRSG-RATE or with 100 pmol of
RNase A at 25C for 15 min in 15 lL of 0.1 m sodium
phosphate buffer (pH 6.5) containing 2 mm dithiothreitol
and 0.5 mm EDTA After addition of 5 lL of 125 mm
Tris⁄ HCl (pH 6.8) containing 15% (v ⁄ v) glycerol and
0.02% (w⁄ v) bromophenol blue, electrophoretic separation
was performed on 10% (w⁄ v) polyacrylamide gels with
40 mm Tris⁄ acetate (pH 8.0) containing 1 mm EDTA as
running buffer The gels were stained with Coomassie
Bril-liant Blue R250, and the bands of the RI–RNase A or RI–
SGRSGRSG-RATE complexes were excised The gel pieces
were incubated in 25 lL of 250 mm Tris⁄ HCl (pH 8.0)
con-taining 5% (w⁄ v) SDS, 50% (v ⁄ v) glycerol, 0.02% (w ⁄ v)
bromophenol blue and 5% (v⁄ v) 2-mercaptoethanol for
15 min at 25C, and analyzed by SDS ⁄ PAGE
Electropho-resis was carried out on a Midget ElectrophoElectropho-resis Unit
(Hoefer, San Francisco, CA, USA) according to Laemmli
[39], using 5% and 12% (w⁄ v) acrylamide for stacking and
separating gels The gels were stained with Coomassie
Bril-liant Blue R250 After destaining, the gels were evaluated
densitometrically at 560 nm with a CD 60 densitometer
(Desaga, Heidelberg, Germany)
Proteolysis
After preincubation of SGRSGRSG-RATE (3.6 lm) in the
absence or presence of RI (7.2 lm) at 25C for 15 min in
15 lL of 50 mm Tris⁄ HCl buffer (pH 8.0) containing 2 mm
dithiothreitol, the reaction was started by addition of 1.5 lL
of trypsin (1· 10)2mgÆmL)1 in 50 mm Tris⁄ HCl buffer,
pH 8.0, containing 10 mm CaCl2) After 15 min (absence of RI) or 1 h (presence of RI) at 25C, the reaction was stopped by addition of 5 lL of phenylmethanesulfonyl fluo-ride (50 mm, dissolved in 2-propanol) The samples were dried under nitrogen and analyzed by SDS⁄ PAGE as described above
Alternatively, SGRSGRSG-RATE was incubated with
RI (1.5 lm each in 200 lL of 100 mm sodium phosphate buffer, pH 6.55, containing 2 mm dithiothreitol) at 25C for 15 min Then, 2 lL of trypsin (0.1 mgÆmL)1 in 50 mm Tris⁄ HCl buffer containing 10 mm CaCl2, pH 8.0) was added After 1 h at 25C, the sample was subjected to cation exchange chromatography on a SOURCE S FPLC system (see above) As references, RNase A and SGRSGRSG-RATE were analyzed in the absence and presence of RI as well Manually collected fractions were analyzed by MALDI MS (Reflex; Bruker-Franzen, Bremen, Germany) after desalting of the protein samples with ZipTip pipette tips (Millipore, Schwalbach, Germany)
Activity assay
For analysis of the catalytic activity of SGRSGRSG-RATE
in comparison with that of RNase A, a fluorometric assay employing the low molecular mass substrate FAM-AUAA-T AMRA was used [40,41] Values of kcat⁄ KMwere determined
in 100 mm 2-(N-morpholino)ethanesulfonic acid-NaOH (pH 6.0) containing 100 mm NaCl SGRSGRSG-RATE (100 pm, corresponding to 200 pm RNase A entities) was incubated in the absence or in the presence of RI (100 pm or
25 nm) for 5 min, and the reaction was started by addition of FAM-AUAA-TAMRA (final concentration 50 nm) After a distinct time interval, 2 lL of trypsin (0.5 mgÆmL)1) was added The reaction was finalized by addition of 1 lL of RNase A (73 lm, for complete cleavage of all substrate) Flu-orescence emission at 515 nm was followed on a Fluoro-Max-2 spectrometer (Jobin Yvon) upon excitation at 490 nm Values of kcat⁄ KMwere determined from the equation
kcat=KM¼ DF
ðFinitial FfinalÞ ½E
where DF is the change in the fluorescence signal of the sam-ple per second, Finitialis the signal after addition of substrate,
Ffinalis the signal after cleavage of all substrate by addition
of RNase A, and [E] is the concentration of enzyme
Acknowledgements
The authors are grateful to R T Raines (University of Wisconsin-Madison, WI, USA) for providing the plas-mid for RI The Land Sachsen-Anhalt is gratefully acknowledged for supporting this work (3537C⁄ 0903T)
A Schierhorn, Martin-Luther University, Halle, Germany, is acknowledged for MS measurements
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Supporting information
The following supplementary material is available: Fig S1 Superimposition of the crystal structures of the RATEs with the linker sequences GPPG, SGSGSG, and SGRSGRSG
Fig S2 Analysis of the stoichiometry of the RIÆSGRSGRSG-RATE complex by native PAGE Fig S3 Analysis of the tryptic cleavage in the RIÆSGRSGRSG-RATE complex by FPLC and MALDI mass spectrometry
Table S1 Crystallographic data processing and refine-ment statistics for the RATEs with the linker sequences GPPG, SGSGSG, and SGRSGRSG
This supplementary material can be found in the online version of this article
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