Computational Enzymology of Ribozymes (from metal-ion to nucleobase catalysis and back ?)

Computational
Enzymology of Ribozymes
from metal-ion to nucleobase catalysis and
back ?
Laboratoire ARN, RNP, structure-fonction-maturation,
Enzymologie Moléculaire et Structurale ( AREMS )
Fabrice Leclerc

Downloaded from cshperspectives.cshlp.org on January 9, 2012 - Published by Cold Spring Harbor Laboratory Press
Schrum et al., 2010; Meierhenrich et al., 2012

J.P. Schrum, T.F. Zhu, and J.W. Szostak
Protocells & RNA

Figure 1. A simple protocell model based on a replicating vesicle for compartmentalization, and a replicating
• containing oligoribonucleotides
genome to encode heritable information. A complex environment provides lipids, nucleotides capable of
biological-like behaviors (MM.

equilibrating across the membrane bilayer, and sources of energy (left), which leads to subsequent replication
of the genetic material and growth of the protocell (middle), and finally protocellular division through
Hanczyc: Phil. Trans. R. Soc. B.

physical and chemical processes (right). (Reproduced from Mansy et al. 2008 and reprinted with permission
from Nature Publishing #2008.)
us to reconstruct plausible pathways and scenar- tial function of creating an internal environment
ios for the origin of life. within which genetic materials can reside and
• from “protocells” with

The term protocell has been used loosely metabolic activities can take place without being
to refer to primitive cells or to the first cells. lost to the environment. Modern cell mem-
Here we will use the term protocell to refer spe- branes are composed of complex mixtures of
cifically to cell-like structures that are spatially amphiphilic molecules such as phospholipids,
delimited by a growing membrane boundary, sterols, and many other lipids as well as diverse

• to “modern” cells
and that contain replicating genetic informa- proteins that perform transport and enzymatic
tion. A protocell differs from a true cell in that functions. Phospholipid membranes are stable
the evolution of genomically encoded advanta- under a wide range of temperature, pH, and

(JW. Szostak)
geous functions has not yet occurred. With a salt concentration conditions. Such membranes
genetic material such as RNA (or perhaps one are extremely good permeability barriers, so that
of many other heteropolymers that could pro- modern cells have complete control over the
vide both heredity and function) and an appro- uptake of nutrients and the export of wastes
priate environment, the continued replication through the specialized channel, pump and

2011, ...)
of a population of protocells will lead inevitably pore proteins embedded in their membranes.
to the spontaneous emergence of new coded A great deal of complex biochemical machinery
functions by the classical mechanism of evolu- is also required to mediate the growth and divi-
tion through variation and natural selection. sion of the cell membrane during the cell cycle.
Once such genomically encoded and therefore The question of how a structurally simple proto-
heritable functions have evolved, we would cell could accomplish these essential membrane

• ...
consider the system to be a complete, living bio- functions is a critical aspect of understanding
logical cell, albeit one much simpler than any the origin of cellular life.
modern cell (Szostak et al. 2001). Vesicles formed by fatty acids have long been
studied as models of protocell membranes
BACKGROUND (Gebicki and Hicks 1973; Hargreaves and
Deamer 1978; Walde et al. 1994a). Fatty acids
Membranes as compartment boundaries

RNA Chemicals
• chemical components of RNA:
nucleobases (Powner et al.,
2010), sugars (Cocinero et al., Reviews U. J. Meierhenrich et al.

2011)
DOI: 10.1002/anie.200905465
The Origin of Life

On the Origin of Primitive Cells: From Nutrient Intake
to Elongation of Encapsulated Nucleotides
Uwe J. Meierhenrich,* Jean-Jacques Filippi, Cornelia Meinert, Pierre Vierling, and
Jason P. Dworkin

• from “primitive” (TNA: H. Yu
Keywords: Dedicated to Professor Wolfram H.-P.
amphiphiles · liposomes · micelles · Thiemann
nucleotides · vesicles

et al., 2012) to “modern” sugar-
phosphate backbones

• ...
• to RNA Angewandte
Chemie

RNA: Information,Catalysis
• from self-replicators (JW
Szostak, 2012) to self-catalysts
and “modern” ribozymes

• from RNA to protein catalysts
• from the RNA to the ‘protein’
world (activation of GTPases:
Bange et al., 2011)

• from RNA to DNA genetic
information (non-coding RNAs
in “modern” genomes)

A short story of ribozymes
self-cleaving self-splicing

Hammerhead
Ribozyme self-cleaving
HDV Ribozyme
self-cleaving
Group I intron Ribozyme
T. Cech, (Nobel Prize, 1986)
self-cleaving
Hairpin Ribozyme
Leadzyme
other natural ribozymes: Group II intron ribozyme, RNase P, ...
artiﬁcial ribozymes (SELEX): amide bonds, methylations, Diels Alder, ...

Self-Splicing / Self-Cleaving
5' 5'
Ni Ni
ribozyme O
O
ribozyme O
O
4' 4'
3' O O 2' H 3' O O 2' H (OH-)
(H2O) (OH-) (H2O) Mg2+
P P
Mg2+ 5'
O OR Mg2+ Mg2+ O OR
Ni+1 OS Ni+1 RO(-)H
5' OS
O2' O 3' O2' O 3' Mg2+
H O R4' H O R4'
SN2(P) reaction
5'
O Ni (“in-line”) 5'
O4' Ni
His12 O
3' O O 2' H Asp424 O4'
5' P
OR B(-) N NH 3' O O 2' H (OH-)
Ni+1 O (H2O) Mg2+
OS P
O 2' O 3' AH(+) Mg2+ O OR
Ni+1 OS RO(-)H
H O R4'
5'
O2' O 3' Mg2+ Glu357
H O R4'
His119
N H RNase A 3’-5’-exonuclease
HN

A prototype for RNA
Catalysis
“Minimum” Hammerhead Ribozyme
substrate/enzyme cleavage site

Canonical 2D Structure folded 2D Structure
Scott, 1999 Scott et al., Science, 1996

One-Metal Ion Models
5'
Ni
NMR model (inactive)
O
O4'
3' O O 2' H
5' P B(-)
O OR
Ni+1
OS
O 2' O 3' AH(+)
H O R4'
630 Nucleic Acids Research, 2005, Vol. 33, No. 2
Mg2+ Ca2+, Mn2+, Co2+, Cd2+
more readily among CN of 4, 5 or 6. It should be noted cobalt
hexamine fails to induce activity with either class II or class IV
ribozymes (data not shown), which is consistent with the inner
sphere coordination of the metal ion.
The characteristics described previously are also related to
the ‘hard’ or ‘soft’ character of metals. In describing metals,
hard generally indicates an electron cloud that is difﬁcult to
deform and has low polarization potential, while soft generally
indicates an easily deformable electron cloud that permits
high polarization and favors bonding interactions of a more
covalent character. Figure 8D plots Z/r versus I2, the second
ionization constant, and is one way of representing hard versus
soft nature (32). It is striking from this ﬁgure that the Class II
switch has a binding pocket that recognizes a cluster of
‘borderline’ metals (and Cd2+ which is considered a soft
metal). This characteristic is perhaps best for explaining the
high level of discrimination against Mg2+, while allowing a
cluster of borderline metals to act as activators. It may be that
relatively small binding motifs can be created, which recogn-
ize metals based on their hard-soft character.
experimental: Dahm & Uhlenbeck, 1993
The commonality of divalent metal binding pockets

Zivarts et al., NAR, 2005
in RNAtheoretical: Torres et al., 2003
The particular type of metal-binding pockets that presumably

5'
Ni
O
O4'
3' O O 2' H
5' P B(-) -HO
O OR
Ni+1 Mg2+
OS
O 2' O 3' AH(+)
H O R4'
Mg2+ Ca2+, Mn2+, Co2+, Cd2+


5'
Ni
O
O4'
3' O O 2' H
5' P B(-) OH2
O OR
Ni+1 Mg2+
OS
O 2' O 3' AH(+)
H O R4'
Mg2+ Ca2+, Mn2+, Co2+, Cd2+


5'
Ni
O
O4'
3' O O 2' H
5' P B(-)
O OR
Ni+1
OS
O 2' O 3' AH(+)
H O R4'
OH2
Mg2+ Mg2+ Ca2+, Mn2+, Co2+, Cd2+


ound water in the fully hydrated La3ϩ ion, the low kobs for

Two-Metal Ion Models
cleavage reaction involving the La3ϩ ion in both positions
not compatible with the observed correlation between the
a of a water bound to a metal ion and the kobs produced by
ferent divalent metal ions. That correlation has been inter-
ted in the metal hydroxide model (Fig. 4) as an effect on
5'
concentration of the aqueous metal hydroxide, which then
O
N
ves as a Brønsted base in the abstraction of the proton from
i
2Ј-oxygen. We have argued (12) that this logic is flawed,
X-ray (active)
O
ause the metal hydroxide complexes4' formed with metal
s with lower pKa values are weaker bases and, therefore,
O O H
uld be less able to abstract3' 2Ј-OH proton, despite their
the
B(-)
ater concentration. This conclusion is supported by the data
P
sented in Fig. 3 because the pKa of the 2Ј-OH is two or more
O Mg 2+
a units higher than those of any of the aqueous metal ions
O R
N
died, making the metal hydroxide poorly suited to the task
i+1
has O
deprotonating the 2Ј-OH. It 5' been convincingly shown
S
O 2' O
t proton transfer does 3' occur in the rate-determining
not 2+
H O
4' R AH(+) Mg
p of the ribozyme cleavage reaction (30). The observed pH
pendence and the correlation between the pKa values of the
ueous metal ions and kobs must, therefore, reflect the effects
Mg2+/La3+

La3+/La3+

experimental: Pontius et al., 1997; Lott et al., 1998
Mg2+/Mg2+ theoretical: Boero et al., 2005; Leclerc & Karplus, 2006

Two-Metal Ion Models
X-ray (active)

20Å

A Specific Metal Ion in the Hammerhead Ribozyme 26823
longest time courses (48 –96 h). Each phase of the time course was
10-fold faster at pH 7.5 than at pH 6.5, as expected if each process
were limited by the chemical step (15). Finally, purification of this
phosphorothioate-substituted HH16 by anion exchange HPLC (8) re-
sulted in partial separation of ribozyme forms such that the two phases
had identical rate constants to those observed in the racemic mixture
but different relative amplitudes (one fraction gave 0.8 of the fast
component and 0.2 of the slow, whereas a second fraction gave 0.2 of the
fast and 0.8 of the slow).
Rates and relative amplitudes of the two phases for reactions in 10
mM Mg2⌅ did not change upon addition of 0.2 mM EDTA or 2 mM
dithiothreitol to the reaction mixture, suggesting that neither kinetic
process depended on the presence of contaminating metal ions. In
reactions with added Cd2⌅, the concentration of EDTA carried over
from the ribozyme and substrate stocks was ⌃15 nM.

experimental: RESULTS
Peracchi et al., 1997 We have used two different hammerhead ribozyme con-
structs, HH⇥1 and HH16 (Scheme 1), in testing the role and

Reaction Path Modeling

Reaction Path Following: B3LYP/6-31+G(d,p)//HF/3-21+G(d)

Contribution of Metals to
Catalysis
Relative Free Energy (kcal/mol)

30 exp
ΔG = 20.1 kcal/mol
25 22.9 kcal/mol
20.8 kcal/mol
20
no metal
15 19.3 kcal/mol 1 metal
10 2 metals
5 dianionic mechanism
0
-5
-10
-15
-20 B3LYP/6-31+G(d,p) Lopez et al., 2006
B3LYP/6-311+G(2d,2p)//B3LYP/6-31G(d,p) Torres et al., 2003
-25 B3LYP/6-31+G(d,p)//HF/3-21+G(d) Leclerc & Karplus, 2006
-30
I II III IV V VI VII VIII IX
Reaction Coordinate

‘Ion Atmosphere’ Model
No Metalloenzyme
5'
O Ni
O
4'
3' O O 2' H
P B(-)
O
5' OR
Ni+1 OS
O2' O 3' AH(+)
H O R4'

Murray et al., Chem. & Biol., 1998
Curtis & Bartel, RNA, 2001
O’Rear et al., RNA, 2001

Minimum and Full-Length
HH ribozymes

Wang et al., Biochem., 1999 Khvorova et al., Nat. Struct. Biol., 2003

de la Peña et al., EMBO J., 2003 Canny et al., JACS, 2004

A Nucleobase
Catalyst X-ray (active)

5' O- H
O C17
O4' H
O6 N7
3' O O 2' H
P N1 N
R O
5' OR G12
OS N
H2N
H
H
O H O
G8 2'

O
O

5'
O experimental: Chi et al., 2008
theoretical: Lee et al., 2008

A Nucleobase
Catalyst X-ray (active)

5' O- H
O C17
O4' H
4'
O6 N7
3' O O 2' H
P N1 N
R O
5' OR G12
OS N
H2N
H
H
O H O
G8 2' Mg2+
O
O

5'
O experimental: Chi et al., 2008
theoretical: Lee et al., 2008

Metal Ions / Nucleobases
as Catalysts
5' 5'
Ni O Ni
O O4'
O4'
3' O O 2' H 3' O O 2' H
AH(+) P B(-)
P O
5' OR
O OR Ni+1
Ni+1 RO(-)H
OS
5' OS O 2' O 3' AH(+)
O 2' O 3' H O R4'
H O R external nucleophile
4' internal nucleophile
HDV, Hairpin,
group-I, group-II introns Hammerhead, etc

Fedor & Williamson, Nat. Rev. Mol. Cell Biol., 2005

Catalytic Strategies in Self-
Cleaving Ribozymes
5'
O Ni
O4'
Hairpin Ribozyme
3' O O 2' H
P B(-) -O
5'
O OR N
Ni+1 OS
O2' O 3' AH(+) N N
H O R N R
4'
N NH2 H2N
G-8
N N H
R N
A-38 Rupert & Ferré d’Amaré, Nature, 2001

Cleaving Ribozymes
5'
O Ni
O4'
Hairpin Ribozyme
3' O O 2' H
P B(-) O
5'
O OR N
Ni+1 OS
O2' O 3' AH(+) H N N
H O R N R
4'
NH2 H2N
N G-8
N N
R N
Salter et al., Biochem., 2006
A-38 Nam et al., RNA, 2008

Cleaving Ribozymes
5'
O Ni
O4'
HDV Ribozyme
3' O O 2' H
P B(-) H2N
O
5' OR
Ni+1 OS
O2' O 3' AH(+) N C-75
H O R N
4' R
O
OH2
2+
Mg

Perrotta et al., NAR, 1999

Cleaving Ribozymes
5'
O Ni
O4'
HDV Ribozyme
3' O O 2' H
P B(-) -HO
O
5' OR
Ni+1 OS Mg2+
O2' O 3' AH(+)
H O R4'
NH2

C-75 N H
N
R O
Nakano & Bevilacqua, JACS, 2001
Liu et al., J. Phys. Chem., 2007

Metal Catalysts in the
Hammerhead Ribozymes ?
5'
O Ni
O O- H
4'
H
3' O O 2' H B(-) O6
P N7
O OR Mg2+
Ni+1 OS N1 N
5' G12
O2' O 3' N
H O R AH(+) et al.
4'
Osborne H2N
e Scheme 1
n
e
r
n
d
+

U
al
z
2 Osborne et al., Biochem., 2009

Metal Binding in Self-
Cleaving/Splicing Ribozymes
A Hammerhead B HDV
C-site C-site

bridging-site bridging-site

Hairpin Group-I
C D
Intron

unable to rescue activity for the A13 or A14 phosphoro- high negative potentia

Metal Binding Sites in the
thioate substitutions (Ruffner & Uhlenbeck, 1990; Knoll also modeled metal b
et al+, 1997; Peracchi et al+, 1997; Scott, 1997)+ The A9 stead of the metal inte
phosphate is part of a metal-binding site observed in posed here (Fig+ 4), the
the original X-ray structure of the hammerhead (Pley with the N1 of G8 + We

Hammerhead Ribozyme ?
et al+, 1994), where a Mn 2ϩ ion is ligated by the pro-R P Brownian-dynamics sim

FIGU
dem
the h
high
ing s
ture
The
meta
phat
to m
phat
gand
resid
colo
illust
and

Chartrand et al., RNA, 1997 Hansen et al., RNA, 2008

2’OH activation ?

O4' O4'

<
OH-
2' 2'
3'O O H OH- 3'O O H OH2
P Mg2+(VI) P Mg2+(VI)
H 3C O OR H3C O OR
5' OS 5' OS
General Base Lewis Acid

Zdenek et al., J. Phys. Chem., 2011

2’OH activation ?
H H
O4' O O
4' H2N
H 2N N H
O 2' H O 2' H

<
3' O 3' O
N N
P N P
5' OR N 5' OR N
H3C O H H3C O O
OS H N OS
O H
N Mg2+(VI) H
O
H OH
O4'
O N
3' O O 2' H
P N N
H3C O
5' OR H
OS N
H2N
Zdenek et al., J. Phys. Chem., 2011

General Acid/Base
Catalysis in RNA cleavage
5'
RNase A Ni
O
O4'
His12
3' O O 2' H
5' P B(-) N NH
O OR
Ni+1
OS
O 2' O 3' AH(+) +H N Lys41
O R 3
H 4'
Phe120-NH-
His119
N H
HN

Raines, Chem. Rev., 1998

Cooperative Models in
Self-Cleaving ?
Ni
RNA5' O 4' H B(-)
O O M/H-R
2'
O
3' P O
O
Ni+1
O
5' O
R-H/M
AH(+) O OH
RNA3'

Cooperative Models in
Self-Cleaving ?
Mg2+
O- N N
C17 -O N R
N N C17
RNA5' O 4' H R G-12
N RNA5' O 4' H N N
O O H2N G-12
2'
O O
O
2' Mg2+ NH2
3' P O O
3' P O
O
O
N1.1
Mg2+ O Mg2+ O
N1.1
5' O 5' O
O H O H
2' O OH 2' O OH
G RNA3' G
O RNA3'
O O O
G-8 RNA3' G-8 RNA3'
O O
RNA5' Leclerc, Molecules, 2010 RNA5'

Metal Ions back in the
and DeRose 2000; Boots et al. 2008). Moderate rates of
catalysis can also be achieved in molar concentrations of
monovalent cations, an important property that helped to
uncover the critical roles of nucleobases in the HHRz re-

Hammerhead Catalysis
action mechanism (Murray et al. 1998; O’Rear et al. 2001;
Bevilacqua et al. 2004). At physiological ionic strengths, the
HHRz requires divalent ions for appreciable rates of catal-
11 - Published by Cold Spring Harbor Laboratory Press divalent
ysis; therefore, it is reasonable to assume that the
metal-dependent channel is the primary mode of catalysis in
nature (Khvorova et al. 2003).
The HHRz was studied for years in its simplest active
form, as three short helices meeting at a junction of con-
served nucleotides that form the active site of the ribozyme
(for review, see Blount and Uhlenbeck 2005). Studies using
this ‘‘truncated’’ form of the HHRz (trHHRz) led to a
model of catalysis in which a catalytic metal in the P9/
G10.1 site coordinates the pro-R oxygen of the scissile
phosphate, presumably to stabilize the negative charge of
the phosphorane transition state (Peracchi et al. 1997;
Wang et al. 1999). Based on detailed metal-rescue exper-
iments, Wang et al. (1999) predicted that the metal ion
coordinates to the P9/G10.1 site in the ground state and
bridges to the scissile phosphate in the transition state of
the trHHRz reaction. A ground state that is very different
from the transition state is consistent with structural
studies of the truncated HHRz, which in general did not
show catalytically relevant atoms within appropriate dis-
tances of the active site (Blount and Uhlenbeck 2005). In
˚
these structures, the P9/G10.1 metal ion site is z20 A away
from its predicted ligand during catalysis, the pro-R oxygen FIGURE 1. (A) Secondary structure of the modified Schistosoma
Ward &DeRose,mansoni HHRz (MSL1L2) (Osborne et al.S. mansoni in these (2OEU)
of the scissile phosphate (Pley et al. 1994; Scott et al. 1995). RNA, 2012 active site of the 2005) used HHRz studies.
(B) Crystallographic

Metal Ions/Nucleobase
Catalysts in the RNA World
ES*≠
Mg2+ (Mg2+ + nucleobases)

ES≠
Mg2+ (Mg2++ nucleobases)

ES*

ES

non-enzymatic catalysis
metal ion catalysis
metal+nucleobase catalysis
EP

Acknowledgments
• Zdenek Chval (University of South Bohemia, CK)
• Daniela Chvalová (University of South Bohemia, CK)
• Xavier Lopez (Euskal Herriko Unibertsitatea, SP)
• Annick Dejaegere (ESBS Strasbourg, France)
• Darrin M. York (Rutgers University, USA)
• Martin Karplus (Harvard University, USA)

Thank you !
G-12

C17

O2’
O3’
O2’
O5’

G-8
N 1.1

Computational Enzymology of Ribozymes (from metal-ion to nucleobase catalysis and back ?)

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