Atom-Specific Mutagenesis Reveals Structural and Catalytic Roles for an Active-Site Adenosine and Hydrated Mg2+ in Pistol Ribozymes

Article abstract of DOI:10.1002/anie.201708679

The pistol RNA motif represents a new class of self-cleaving ribozymes of yet unknown biological function. Our recent crystal structure of a pre-catalytic state of this RNA shows guanosine G40 and adenosine A32 close to the G53–U54 cleavage site. While the N1 of G40 is within 3.4 ? of the modeled G53 2′-OH group that attacks the scissile phosphate, thus suggesting a direct role in general acid–base catalysis, the function of A32 is less clear. We present evidence from atom-specific mutagenesis that neither the N1 nor N3 base positions of A32 are involved in catalysis. By contrast, the ribose 2′-OH of A32 seems crucial for the proper positioning of G40 through a H-bond network that involves G42 as a bridging unit between A32 and G40. We also found that disruption of the inner-sphere coordination of the active-site Mg²⁺ cation to N7 of G33 makes the ribozyme drastically slower. A mechanistic proposal is suggested, with A32 playing a structural role and hydrated Mg²⁺ playing a catalytic role in cleavage.

Full text of DOI:10.1002/anie.201708679

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Accepted Article
Title: Atom-specific mutagenesis reveals structural and catalytic roles
for active site-adenosine and hydrated Mg2+ in pistol ribozyme
Authors: Sandro Neuner, Christoph Falschlunger, Elisabeth Fuchs,
Maximilian Himmelstoss, Aiming Ren, Dinshaw Patel, and
Ronald Micura
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201708679
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Atom-specific mutagenesis reveals structural and catalytic roles
for active site–adenosine and hydrated Mg2+ in pistol ribozyme
Sandro Neuner,+ Christoph Falschlunger,+ Elisabeth Fuchs, Maximilian Himmelstoss, Aiming Ren,
Dinshaw J. Patel, and Ronald Micura*
Abstract: The pistol RNA motif represents a new class of self-
cleaving ribozymes of yet unknown biological function. Our recent
crystal structure of a pre-catalytic state of this RNA shows guanosine
G40 and adenosine A32 close to the G53-U54 cleavage step. While
the N1 of G40 is in 3.4 Å distance to the modeled G53 2’-OH group
that attacks the scissile phosphate and hence suggests its direct role
in general acid–base catalysis, the function of A32 is not clear. We
present evidence by atom-specific mutagenesis that neither N1 nor
N3 base positions of A32 are involved in catalysis. By contrast, the
ribose 2’-OH of A32 seems crucial for proper positioning of G40 via
H-bond networks that involve G42 as a bridging unit between A32
and G40. We also found that disruption of the inner-sphere
coordination of the active site–Mg2+ cation to N7 of G33 makes the
ribozyme drastically slower. A mechanistic proposal is suggested
with A32 playing a structural and hydrated Mg2+ playing a catalytic
role in cleavage.
The discovery of twister, twister sister, pistol and hatchet self-
cleaving ribozymes by comparative genomic analysis has
reinvigorated research to explore the chemical strategies RNA
utilizes to catalyze chemical reactions.[1,2] To this end, the three-
dimensional architectures of twister,[3-6] twister-sister,[7,8] and
pistol ribozymes[9,10] in their pre-catalytic states have been
recently revealed by crystallography; these provide a solid
foundation for mechanistic proposals that await their
experimental evaluation in solution by structure-function analysis
and targeted atomic mutagenesis. While for twister ribozymes,
chemical,[4,6,11,12] biochemical,[1-3,13] and computational[14] studies
Figure 1. Secondary structure of the env25 pistol ribozyme (a) and crystal
structure of the active site showing the dG53-U54 cleavage step (yellow) and
have already provided valuable insights into the molecular
mechanism of this ribozyme,[15,16] such studies lag behind for the
three other new ribozyme classes.
the closest neighbors G40 and A32 (pdb code 5K7C)[9] (b). Numbers in grey
indicate distances; red arrow indicates the direction of nucleophilic attack on
the scissile phosphate (from the 2’-O of G53 in the native ribozyme); for ideal
in-line alignment of the 2’-O relative to the to-be-cleaved P–5’O bond, the
angle t is 180°. (c) RNA phosphodiester cleavage: The internucleotide linkage
(‘scissile’ phosphate) passes through a pentacoordinate transition state that
results in two cleavage products carrying either a 2’,3’-cyclic phosphate
terminus and a 5’-hydroxyl terminus, respectively. The four catalytic strategies
that impact on the reaction are:[23] α, in-line nucleophilic attack, SN2-type
(blue); β, neutralization of the (developing) negative charge on nonbridging
phosphate oxygens (purple); γ, deprotonation of the 2’-hydroxyl group (red);
and δ, neutralization of negative charge on the 5’-oxygen atom by protonation
(green).
[*]
S. Neuner,[+] C. Falschlunger,[+] E. Fuchs, M. Himmelstoss, Prof. R.
Micura
Institute of Organic Chemistry and Center for Molecular Biosciences
(CMBI)
Leopold-Franzens University
Innrain 80-82, 6020 Innsbruck, Austria
E-mail: ronald.micura@uibk.ac.at
Prof. D. J. Patel
Structural Biology Program, Memorial Sloan-Kettering Cancer
Center
New York, New York 10065, USA
Dr. A. Ren
Life Science Institute, Zhejiang University
Hangzhou 310058, China
Thus far, pre-catalytic structures of the pistol ribozyme
have been reported from two groups,[9,10] with good consensus
on the overall folding topology and the alignment of catalytic
residues at the cleavage site (Figure 1). Both pyrimidine and
purine at the pyrimidine-purine cleavage step of the pistol
ribozyme are anchored in a splayed-apart conformation with in-
line alignment of the modeled 2′-O of the purine nucleoside
properly located for attack on the adjacent to-be-cleaved P-O5′
[+]
These authors contributed equally.
Supporting information for this article is given via a link at the end of
the document.
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bond (Figure 1b,c). Importantly, highly conserved residues, a
guanosine (G40; N1 position)[9,10] and a purine nucleoside (either
A[9] or G[10]) (position 32; N3 and 2′-OH) are aligned to potentially
act as general base and general acid, respectively (Figure 1b). A
first set of cleavage assays with ribozyme mutants was
supportive for these putative roles although an evaluation via
deazapurine ribonucleosides has not yet been provided.[9]
Interestingly, NMR spectroscopy revealed only a slightly
increased pKa of 4.7 for A32 for a non-cleavable (dG53
modified) pistol variant.[9] Furthermore, our previous studies
demonstrated that the deletion of the 2’-OH alone (2’-
deoxyadenosine) significantly reduced the rate of cleavage while
simultaneous deletion of N3 and 2’-OH (3-deaza-2’-
deoxyadenosine, c3dA32) abolished activity.[9] The precise roles
of these functional groups, however, remained unclear, not least
because the key mutations of 1-deazaadenosine (c1A) and 3-
deazaadenosine (c3A) both comprising the native 2’-OH group
were not included in the previous studies.[9,10]
that are not commercially available. Therefore, the c1A
phosphoramidite was synthesized in eleven steps in analogy to
previously published procedures (Supporting Information),[17]
and c3A phosphoramidite was synthesized in five steps
according to our recent protocol.[18] With these phosphoramidites
in hand, we synthesized the corresponding two-stranded pistol
ribozymes with c1A and c3A mutations in position 32. Next, we
performed cleavage assays of these two ribozyme variants
using anion-exchange high-performance liquid chromatography
(HPLC; Figure 2). Under single-turnover conditions, wild-type
env25 pistol ribozyme cleaves its substrate to completeness in
less than 10 min in the presence of 2 mM Mg2+ and 100 mM K+
at pH 7.5 (30 mM HEPES buffer) and at 23°C (Figure 2a). When
A32 was mutated to c1A, cleavage appeared as fast as for the
wild-type ribozyme (Figure 2b); this was surprising because N1
is the preferred site of protonation of adenosine and the prime
candidate for general acid catalysis to donate a proton to the 5’-
O leaving group.[24] However, we remembered that our studies
on twister ribozymes showed that only the c3A but not the c1A
twister variant was deleterious for cleavage.[15] Therefore, we
decided to next analyze the c3A32 pistol variant. Even more
surprising, we observed that for this c3A32 variant, cleavage was
absolutely comparable in activity to the wild-type ribozyme
(Figure 2a,c).
We have therefore set out to shed light on the putative
mechanistic role of A32 by applying an atomic mutagenesis
approach that comprehensively utilizes deazapurine ribo-
nucleosides. Because the imino N-hydrogen donor/acceptor
capabilities become lost in site-specific manner if c1A and c3A,
respectively, were substituted for A32 in pistol ribozymes, their
cleavage activities constitute
a reliable measure for the
verification/elimination of this nucleobase to participate in
general acid–base catalysis for phosphodiester cleavage.
Figure 3. Self-cleavage of the env25 pistol ribozyme and mutants. (a) 2-
Aminopurine fluorescence assays using a A57Ap variant. (b) Exemplary
fluorescence time courses for rate determination at 15 °C; arbitrary units (a.u);
MgCl2 was added at t = 0; conditions: c(RNA) = 0.5 μM (1:1 ratio), 50 mM
KMOPS, 100 mM KCl, 15°C, pH 7.5; mixing was performed manually in less
than 2 s resulting in 10 mM Mg2+ concentration (each kobs obtained from three
independent experiments).
Figure 2. Self-cleavage of the env25 pistol ribozyme (a) and mutants (b-f).
Cleavage analysed at 55 µM RNA each strand; 2 mM MgCl2, 100 mM KCl, 30
mM HEPES, pH 7.5, 23 °C. R 47-nt ribozyme, S 11-nt substrate; C1 and C2,
5-nt and 6-nt cleavage products. HPLC conditions: Dionex DNAPac column
(4x250 mm), 80 °C, 1 ml min-1, 0–60% buffer B in 45 min. Buffer A: Tris–HCl
(25 mM), urea (6 M), pH 8.0. Buffer B: Tris–HCl (25 mM), urea (6 M), NaClO4
(0.5 M), pH 8.0.
To additionally verify our findings, we developed a 8-step
synthesis for 1,3-dideazaadenosine (c1c3A) amidite (Supporting
Information; in analogy to a report by Müller et al. for c1c3dA),[19]
The above mentioned deazaadenosine mutations require
phosphoramidite building blocks for RNA solid-phase synthesis
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synthesized the corresponding c1c3A32 modified pistol ribozyme
and tested its activity. We again found unhindered specific
phosphodiester cleavage (Figure 2d). This confirms that neither
N1 nor N3 can pitch in for its counterpart and that indeed none
of these imino functionalities is involved in general acid base
catalysis.
2’-OH of A32 with most likely lowered pKa (because of the H-
bond to NH2 of G42) might be involved in proton transfer to the
U54 5’O leaving group, although further conformational changes
were needed to bring these groups closer together. At this point
we mention that we also tested A32G,[9] A32dG and A32Gm
(Supporting Figure 1b-c, Supporting Figure 2), resulting in the
same reactivity profiles as observed for A32, A32dA and A32Am,
highlighting the consistency of our model.
In this context, the precise rates kobs of A32, c1A32, c3A32
and c1c3A32 modified pistol ribozymes were determined in
comparative manner utilizing
a previously established 2-
aminopurine fluorescence assay (Figure 3a).[25] Indeed, the rates
of the deazaadenosine modified ribozymes are similar (or even
slightly faster) compared to the wild-type ribozyme (Figure 3b-d,
Supporting Table 1).
Because of these unanticipated results, we reanalyzed the
active site residues and focused on the shortest (and most likely
strongest) H-bond interaction of A32, formed between its 2’-OH
to the exocyclic NH2 group of G42 (Figure 4). Equally important,
the O6 of G42 is in 2.7 Å H-bond distance to the NH2 of G53 (of
the cleavage step) and in 2.8 Å H-bond distance to the NH2 of
G40, the latter assigned to take the role as general base in pistol
ribozyme cleavage. It seems that G42 is the structural ‘glue’
between G40 (general base) and A32 (putative general acid)
and thereby generates a cleft to accommodate the scissile
phosphate of the splayed-apart G53-U54 cleavage step.
Figure 5. Self-cleavage of 2’-OH versus 2’-NH2 A32 modified env25 pistol
ribozyme. Cleavage analysed at 55 µM RNA each strand; 2 mM MgCl2, 100
mM KCl, 30 mM HEPES, pH 7.0 or 7.5 or 8.5 (c-d), 23 °C. For pH 6.0 (b),
MES buffer (30 mM) was used; R 47-nt ribozyme, S 11-nt substrate; C1 and
C2, 5-nt and 6-nt cleavage products. HPLC conditions as in Figure 2, except
60°C column oven temperature.
To learn more about the role of the 2’-OH of A32, we were
wondering how its substitution to an amino group would affect
cleavage activity. We synthesized the corresponding 2’-amino-
2’-deoxyadenosine phosphoramidite,[26] prepared the 2’-NH2–
A32 modified pistol ribozyme and performed cleavage
experiments. It was reported previously that the cleavage rate
kobs of the pistol ribozyme increases with increasing pH (albeit
measured at suboptimal 25 µM Mg2+ concentrations).[20] This
trend was also observed in our experimental set-up at saturating
2 mM Mg2+ concentrations (Supporting Figure 3). In direct
comparison of 2’-OH versus 2’-NH2–A32 pistol ribozymes
(Figure 5), we found that the native ribozyme cleaves readily at
pH 6.0 and 7.0 while for its 2’-NH2 counterpart, hardly any
cleavage occurs after 10 min (Figure 5b,c). Only at higher pH
values (7.5 and 8.5), the 2’-NH2 modified ribozymes are able to
cleave their substrates almost as efficiently (Figure 5d,e).
Figure 4. Active site of the env25 pistol ribozyme with G42 interactions
highlighted (pdb code 5K7C)[9] Nucleoside positions are color-coded: cleavage
step dG53–U54 (yellow); numbers in grey indicate distances in Å; red arrow
indicates the scissile phosphate.
From our previous study, we knew that the A32dA
modified pistol ribozyme showed only about 25% cleavage yield
after 10 min and about 90% after 45 min.[9] In the light of the new
analysis, we additionally tested a A32Am variant and observed
hardly any cleavage after 10 min (Figure 2f) and only miniscule
amounts of cleavage products (about 10%) after 45 min
(Supporting Figure 1a). This is consistent with the loss of the
specific H-bond that otherwise helps to keep G42 and G40 in
place, thereby supporting the architecture for proper
accommodation of the G53-U54 cleavage step. Additionally, the
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The pKa of a 2’-OH group in RNA is assumed to be
approximately 12,[21] while for 2’-NH2 modified pyrimidine
nucleosides[22] in RNA the pKa was determined to be 6 (this
more difficult to verify or nullify (because of the structural
entanglement described above) is the role or requirement of A32
in proton transfer to the 5’-O leaving group (d catalysis; see also
Figure 1c and caption). In its absence (dA32 mutant), the
ribozyme can still cleave, albeit with a significantly reduced rate
(more than 100-fold).[9] It could be concluded that for dA32
modified ribozymes, the purine N3 might in part take over this
role, because only the double deletion of 2’-OH and N3
abolishes cleavage completely.[9] Alternatively, the H+ needed for
protonation of the 5’-O leaving group may stem from the
hydrated Mg2+ that is inner sphere coordinated to N7 of G33 and
in about 5 Å distance to the 5’-O atom. This hydrated Mg2+ is a
candidate for proton transfer given that the 2’-OH of purine-32 is
also not ideally positioned (in ~5.5 Å distance), and additional
conformational changes from the pre-catalytic conformation to
the transition state are required in any case. To finally shed light
on the putative involvement of this hydrated Mg2+ ion, we
synthesized a 7-deazaguanosine–33 (c7G33) pistol ribozyme
that lacks the ability to coordinate to Mg2+ because of the N7-to-
C7 replacement. Strikingly, this modified ribozyme had hardly
any cleavage activity (Figure 6, Supporting Figure 4; kobs = 4.27±
0.4 x 10-3 min-1). This finding suggests a crucial role for the
active site–hydrated Mg2+ that is seen in both crystal
structures.[9,10] Hence, taken all our atomic-mutagenesis
experiments together, this leads to the mechanistic proposal
illustrated in Figure 7.
+
value refers to the corresponding 2'-NH3 moiety). Although the
pKa can be shifted to some extent by the nature of the
nucleobase and its specific RNA micro-environment, it is
reasonable to assume that at pH 6.0 a significant portion of the
ribose (sp3 hybridized) 2’-NH2 is protonated. In its protonated
form, however, it cannot function as H-bond acceptor and
hydrogen bond formation to the 2-NH2 of G42 (sp2 hybridized; H-
bond donor) becomes impaired. As a consequence, proper
formation of the G40–G42–A32 scaffold, which is required to
optimally mould the active site architecture, is compromised
(Figure 4). This explanation is consistent with the observation
+
that no cleavage was observed at pH 6.0 and 7.0 (2’-NH3 A32)
while at higher pH values (>7.0; 2’-NH2 A32) cleavage is
restored (Figure 5).
Figure 6. Active site of the env25 pistol ribozyme with the Mg2+–N7-G33
interaction highlighted (a);[9] note that the hydrated Mg2+ ion is in proper
distance to potentially form a water-mediated hydrogen bond to the non-
bridging oxygen of the scissile phosphate; nucleoside positions are color-
coded as in Figure 1; numbers in grey indicate distances in Å; red arrow
indicates the scissile phosphate; (b) Cleavage of the c7G33 modified pistol
ribozyme is almost completely abolished; setup of cleavage assays and HPLC
conditions as in Figure 5.
Figure 7. Mechanistic proposal for phosphodiester cleavage in the env25
pistol ribozyme.
What can we learn from the current set of experiments
about the mechanism of phosphodiester cleavage for the pistol
ribozyme? In our opinion, the major insight is that neither N1 nor
N3 of the highly conserved purine base (A or G) in position 32 is
directly involved in catalysis in the native RNA. Second, the
structure–sculpting role of A32 has been revealed: A32
participates through its 2’-O···H2N-G42 hydrogen bond in the H-
bond network between G40, G42 and A32 to generate the cleft
for accommodation of the G53-U54 cleavage step; in other
words, A32 assists to position the general base G40. What is
In summary, our experiments shed light on the structural
role of the 2’-OH of the purine nucleoside–32 and of the catalytic
role of the hydrated Mg2+ coordinated to G33 of the pistol
ribozyme. Our preferred view for pistol ribozyme phosphodiester
cleavage draws a mechanistic scenario where a combination of
catalytic strategies is employed. Both available crystal structures
(from independent groups)[9,10] suggest that in the precatalytic
state, the attacking 2’-OH of G53 is orientated nearly ideal for in-
line attack at the scissile phosphate (a catalysis). Likewise, in
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Entry for the Table of Contents
COMMUNICATION
The ribose of a conserved
S. Neuner, C. Falschlunger, E. Fuchs,
M. Himmelstoss, A. Ren, D. J. Patel,
and R. Micura*
adenosine and a hydrated Mg2+
ion in the active site of the pistol
ribozyme contribute the activity-
determining functional groups. Their
potential roles in general acid-base
catalysis versus active site
Page No. – Page No.
Atom-specific mutagenesis reveals
structural and catalytic roles for
active site–adenosine and hydrated
Mg2+ in pistol ribozyme
structuring is discussed.
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