Structural Relationships Among the Ribosomal Stalk Proteins from

Structural Relationships Among the Ribosomal Stalk Proteins from

J Mol Evol (2008) 67:154–167
DOI 10.1007/s00239-008-9132-2
Structural Relationships Among the Ribosomal Stalk Proteins
from the Three Domains of Life
Przemysław Grela Æ Pau Bernadó Æ Dmitri Svergun Æ Jan Kwiatowski Æ
Dariusz Abramczyk Æ Nikodem Grankowski Æ Marek Tchórzewski
Received: 14 December 2007 / Accepted: 9 June 2008 / Published online: 9 July 2008
Ó Springer Science+Business Media, LLC 2008
Abstract The GTPase center of the large ribosomal
subunit, being a landing platform for translation factors,
and regarded as one of the oldest structures in the ribosome, is a universally conserved structure in all domains of
life. It is thought that this structure could be responsible for
the major breakthrough on the way to the RNA/protein
world, because its appearance would have dramatically
increased the rate and accuracy of protein synthesis. The
major part of this center is recognized as a distinct structural entity, called the stalk. The main functional part of the
stalk in all domains of life is composed of small L12/P
proteins, which are believed to form an evolutionarily
P. Grela D. Abramczyk N. Grankowski M. Tchórzewski (&)
Department of Molecular Biology, Institute of Microbiology and
Biotechnology, Maria Curie-Skłodowska University, Akademicka
19, 20-033 Lublin, Poland
e-mail: [email protected]
P. Bernadó
Institut de Recerca Biomèdica, Parc Cientı́fic de Barcelona,
Josep Samitier 1-5, 08028 Barcelona, Spain
D. Svergun
European Molecular Biology Laboratory, Hamburg Outstation,
Notkestrasse 85, 22603 Hamburg, Germany
D. Svergun
Institute of Crystallography, Russian Academy of Sciences,
Leninsky pr. 59, 117333 Moscow, Russia
J. Kwiatowski
Department of Plant Systematics and Geography,
Institute of Botany, University of Warsaw, Al. Ujazdowskie 4,
00-478 Warsaw, Poland
J. Kwiatowski
Department of Ecology and Evolutionary Biology,
University of California, Irvine, CA 92697, USA
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conserved group. However, some data indicate that the
bacterial and archaeo/eukaryal proteins are not related to
each other structurally, and only a functional relationship
may be clearly recognized. To clarify this point, we performed a comprehensive comparative analysis of the L12/P
proteins from the three domains of life. The results show
that bacterial and archaeo/eukaryal L12/P-proteins are not
structurally related and, therefore, might not be linked
evolutionarily either. Consequently, these proteins should
be regarded as analogous rather than homologous systems
and probably appeared on the ribosomal particle in two
independent events in the course of evolution.
Keywords Acidic ribosomal P proteins Ribosomal stalk Ribosome Protein synthesis
Introduction
The ribosome is the core of the translation machinery of all
organisms and assures two key functions: the decoding of
the genetic information and the formation of peptide bonds.
The ribosome, probably being a reminiscence of the
ancient RNA world, is perceived as a ribozyme because its
two primary key functions are dependent on rRNA (Nissen
et al. 2000). However, as (Noller 2004) stated, ‘‘The
invention of protein synthesis, probably instructed and
catalyzed by RNA, was not to create a protein world, but to
extend the structural, and therefore the functional, capabilities of the RNA world, and initially was the crowning
achievement of the RNA world, but also began its demise.’’
It is thought that at a very early stage of life everything
turned on the evolution of translation (Woese 1998). Initially, only small proteins could evolve—along with any
larger, imprecisely translated ones (called ‘‘statistical
J Mol Evol (2008) 67:154–167
proteins’’) that the primitive cell was able to produce and
use (Woese and Fox 1977). Each slight improvement in
that process would have permitted a new generation of
proteins to emerge; these new proteins, in turn, refined and
developed the metabolic pathways and generally improved
the performance of the cell, which then set the stage for a
further round of improvement in translation (Woese 1998).
According to Woese (1998), ‘‘This iterative, bootstrapping
evolution continued until the accuracy of translation
reached a level where it no longer prevented the evolution
of the types of proteins we see today.’’ Therefore, translation was among the first, if not the very first, of the
cellular subsystems to crystallize, and moreover, the
improvement of accurate protein synthesis paved the way
from progenotes to the modern organisms called genotes
(Woese 1998). One of the ribosomal structures responsible
for a major breakthrough on the way from RNA to protein
world could have been the GTPase center, whose appearance, together with translation factors, would have
dramatically increased the rate and accuracy of protein
synthesis, leading to today’s richness of life forms (Frank
et al. 2007; Hury et al. 2006). This ribosomal center, being
an interacting place for translation factors, is regarded as
one of the oldest in the ribosome, and occurs in all living
organisms (Mears et al. 2002). The major part of this
center is recognized on the large ribosomal subunit as a
distinct structural entity, called the stalk. It is composed of
conserved proteins which form an oligomeric structure, and
represents an isolated entity without extensive contacts
with other ribosomal components (Diaconu et al. 2005;
Gonzalo and Reboud 2003; Wahl and Moller 2002). These
proteins exist in two main classes—bacterial and archaeo/
eukaryal. In Bacteria the stalk is composed of two types of
proteins. The first is represented by the L10 protein, which
forms the base of the stalk (Wahl and Moller 2002). L10
proteins from all organisms were found to be similar
(Shimmin et al. 1989) and are classified among the 29
universally conserved proteins constituting the genetic core
of the hypothetical universal ancestor (Harris et al. 2003).
The L12 protein represents the second group (also denoted
L7/L12 in the case of E. coli) and occurs on the ribosome
in multiple copies, attached to it through the L10 protein. A
species-dependent variability in stoichiometry has been
documented for the L12 protein, showing a lack of uniformity of bacterial ribosomes in this respect. In
thermophiles, there are three, whereas mesophiles have two
L12 dimers, forming heptameric L10(L12)6 or pentameric
L10(L12)4 complexes, respectively (Diaconu et al. 2005;
Ilag et al. 2005). The archaeo/eukaryal stalk is also composed of two types of proteins, P0 and P1/P2 (Ballesta and
Remacha 1996; Tchorzewski 2002). P0 is homologous to
the bacterial L10 protein and, forming the base of the stalk,
constitutes the anchor for the two P1/P2 protein dimers
155
(Wahl and Moller 2002). The P1/P2 group is regarded as a
functional counterpart of the bacterial L12 protein, however, it seems that they have no significant similarity in the
primary structure to the bacterial protein (Liao and Dennis
1994). Alignment of P1 and P2 with the archaeal homologue, the aL12 protein, indicated that P1 and P2 form a
distinct monophyletic group and arose from duplication
and subsequent divergence of an ancestral form of archaeal
aL12 that occurred very early in the eukaryotic lineage
(Liao and Dennis 1994). These proteins form P1-P2 heterodimers (Gonzalo et al. 2001; Shimizu et al. 2002;
Tchorzewski et al. 2000b), which in some eukaryotes, such
as the yeast S. cerevisiae, form two distinct P1A-P2B and
P1B-P2A complexes (Guarinos et al. 2001; Tchorzewski
et al. 2003). In all organisms, the stalk is directly involved
in ensuring the speed and accuracy of protein synthesis and
constitutes part of the GTPase-associated center, with
the L12/P proteins being its main functional elements
(Rodnina and Wintermeyer 2001). The whole stalk is
indispensable for cell survival (Krokowski et al. 2006), but
for proper functioning of the ribosome only one dimer is
sufficient in bacteria (Griaznova and Traut 2000), while in
Eukaryotes the P1/P2 proteins are not absolutely required
for the ribosome activity or cell viability (Remacha et al.
1995). Despite the apparent identical function of the L12/P
proteins on the ribosome, they are not interchangeable
between Bacteria and Archaea/Eukaryotes, and moreover,
the P proteins confer factor binding specificity on the
ribosome (Nomura et al. 2006; Uchiumi et al. 1999). What
is more, in Eukaryotes, the P proteins from distinct species
are not fully complementary in terms of function, and it
seems that they have diverged during ribosome evolution
and, apart from their primary function, might also represent
regulatory elements enabling distinct organisms to
accommodate their metabolism to changing environmental
conditions (Rodriguez-Gabriel et al. 2000).
A structural model of the whole stalk has been proposed
for the bacterial ribosome (Diaconu et al. 2005), but the
intact archaeo/eukaryal stalk has not been described yet.
The only structure available today is a low-resolution model
of the S. cerevisiae P1A-P2B dimer, and preliminary analysis showed that the yeast complex does not resemble, in its
shape, the structure of the bacterial counterpart (Grela et al.
2007). Moreover, the evolutionary relationship among the
L12/P proteins from diverse organisms is unclear (Liao and
Dennis 1994). Bacterial L12 proteins were classified as
universally conserved in the three phylogenetic domains
(Mears et al. 2002), but it seems that they are different in
terms of structural organization from their archaeo/eukaryal
counterparts. Therefore the question arises into which
pathway of evolution the L12/P proteins may fall: divergent, where all proteins are coming from a common
ancestor but the rate of evolution was so high that they
123
156
have diverged to the point that no similarity can be detected
anymore; or convergent, where bacterial and archaeo/
eukaryal proteins were recruited independently for the same
function.
Therefore, to shed more light on the evolutionary relationship among the stalk L12/P proteins, we undertook
structural characterization of these proteins from the three
phylogenetic domains: Bacteria, Escherichia coli; Archaea,
Sulfolobus solfataricus; and Eukaryotes, Homo sapiens.
The analyses showed that, considering all structural
aspects, the bacterial and archaeo/eukaryal proteins are not
structurally related, raising the question of the evolutionary
origins of these proteins. In our view the structural data are
more consistent with convergent evolution.
J Mol Evol (2008) 67:154–167
Markov chains were run, with 1 million generations per
chain, and the 50% majority rule consensus tree was built,
with the first 2000 trees discarded. The tree was drawn with
Tree View, Version 1.6.1, for Microsoft Windows (Page
1996). The neighbor-joining (NJ) and minimal evolution
(ME) phylogenetic trees were constructed with the MEGA
program, version 3.1 (Kumar et al. 2004). For both methods, the parameters p-distance model and pairwise deletion
were selected, and the interior branch test and bootstrap
test were performed with 1000 replications each.
Analysis of Secondary Structure Propensity
Materials and Methods
The whole amino acid sequences of the acidic ribosomal
aL12/P proteins were analyzed using secondary structure
prediction tools: Jpred (Cuff et al. 1998) and Sspro (Cheng
et al. 2005).
Protein Expression and Purification
Small-Angle X-Ray Scattering (SAXS) Experiment
The human P1-P2 heterodimer and P2-P2 homodimer were
prepared according to the procedure described earlier
(Grela et al. 2008; Tchorzewski et al. 2000a). Recombinant Sulfolobus solfataricus aL12 protein was prepared as
follows. Appropriate sequence was PCR amplified from
genomic DNA (a kind gift from Dr. Emmanuele De Vendittis, Universita di Napoli Federico II, Italy) with the aid
of specific primers. The amplified fragment was introduced
into expression vector pLM1 (Sodeoka et al. 1993). The
aL12 protein was expressed with a 6xHis-tag on its Nterminus in E. coli BL21(DE3) cells and purified using
affinity chromatography on an Ni column, followed by
size-exclusion chromatography. The bacterial Escherichia
coli L7/L12 protein was expressed using plasmid pET24b
(a kind gift from Dr. Suparna Sanyal, Biomedical Center,
Uppsala, Sweden), and the recombinant protein was prepared according to the procedure published earlier (Mulder
et al. 2004).
The SAXS method was used to determine the threedimensional (3D) structure of the studied proteins. This
method has proven to be a very useful technique for
obtaining low-resolution molecular shape of proteins and
their complexes in solution. The main advantage of solution scattering is its ability to study the structure of native
particles under nearly physiological conditions. Recently,
there has been significant progress in the development of
ab initio methods for low-resolution shape restoration,
placing SAXS as a very fast and reliable structural
approach (Svergun et al. 2001).
The synchrotron radiation X-ray scattering data were
collected in two experimental sessions on the X33 camera
(Boulin et al. 1988; Koch and Bordas 1983) at the European Molecular Biology Laboratory Hamburg (EMBL) on
the storage ring DORIS III of the Deutsches Elektronen
Synchrotron (DESY) using multiwire proportional chambers with delay line readout (Gabriel and Dauvergne 1982).
The scattering patterns from all proteins were recorded at
multiple solute concentrations ranging from 2 to 15 mg/ml
(in the case of L7/L12 protein, from 2 to 50 mg/ml) at a
sample-detector distance of 2.3 m, covering the range of
momentum transfer 0.15 \ s \ 3.5 nm-1 (s = 4p sin(h)/k,
where 2h is the scattering angle and k = 0.15 nm is the Xray wavelength). The data collected in 10 successive 1-min
frames were analyzed for the absence of radiation damage,
averaged after normalization to the intensity of the incident
beam, corrected for the detector response, and the scattering of the buffer was subtracted using the program
PRIMUS (Konarev et al. 2003). The difference curves
were scaled for the protein concentrations and extrapolated
to infinite dilution following standard procedures (Feigin
and Svergun 1987). The maximum dimensions of all
Phylogenetic Analysis
The alignment of amino acid sequences from representative species, obtained using ClustalX1.8 with default
options (Thompson et al. 1997), was manually checked and
edited. For phylogenetic analysis sites of uncertain
homology, which could not be unambiguously aligned,
were removed. After removal, 75 positions (of 105) were
left in the alignment. Bayesian phylogenetic analysis was
performed, and its substitution model with fixed rate
matrices and rate parameters (Prset aamodelpr=mixed,
LSET rates=invgamma) were estimated by the MrBayes
3.0B4 program (Huelsenbeck and Ronquist 2001). Gamma
distribution was approximated using four categories. Four
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J Mol Evol (2008) 67:154–167
proteins Dmax were estimated using the orthogonal expansion program ORTOGNOM (Svergun 1993). The forward
scattering values I(0) and the radii of gyration Rg were
evaluated using the Guinier approximation (Guinier 1939).
These parameters were also computed from the entire
scattering patterns using the indirect transform package
GNOM (Svergun 1992), which also provides the distance
distribution functions p(r) of the particles.
Low-resolution models of the proteins were generated
ab initio by the program GASBOR (Svergun et al. 2001) as
previously described (Grela et al. 2007). The models of
each protein were reconstructed using appropriate numbers
of residues, according to the protein sequence (P1-P2, 229
residues; P2-P2, 230 residues; L7/L12, 242 residues; aL12,
222 residues). For the homodimeric P2-P2 complex,
reconstructions without and also with symmetry restrictions (i.e., assuming P2 point symmetry) were performed.
For each solute, results from a dozen separate GASBOR
runs were averaged to determine common structural features using the programs DAMAVER (Volkov and
Svergun 2003) and SUPCOMB (Kozin and Svergun 2001).
The latter program aligns two arbitrary low- or high-resolution models represented by ensembles of points by
minimizing a dissimilarity measure called normalized
spatial discrepancy (NSD). For every point (in this case,
coordinate of DRs) in the first model, the minimum value
among the distances between this point and all points in the
second model is found, and the same is done for the points
in the second model. These distances are added and normalized against the average distances between the
neighboring points for the two models. Generally, NSD
values close to unity indicate that the two models are
similar. The program DAMAVER generates the average
model of the set of superimposed structures and also
specifies the most typical model (i.e., that having the
lowest average NSD with all the other models of the set).
Rigid Body Modeling
Rigid body modeling represents the method where it is
possible to build models of protein complexes from highresolution structures of their subunits or domains against
low-resolution models of the whole complexes derived
from SAXS data (Petoukhov and Svergun 2005). Molecular modeling was done with the available atomic models
of both domains of the L7/L12 protein. The NMR
(Bocharov et al. 2004) (1RQU; fragment 1-31) and the
crystallographic (Leijonmarck et al. 1980) (1CTF) structures were used as models of the N- and the C-terminal
folded domains, respectively. The program BUNCH
(Petoukhov and Svergun 2005) was employed to model the
protein structure fitting the SAXS merged profile covering
an angular range from 0.014 to 0.34 Å-1. A P2 internal
157
symmetry, found for the N-terminal domain in crystallographic studies (Wahl et al. 2000), was imposed. BUNCH
combines rigid-body with ab initio modeling for proteins
consisting of folded domains, for which high-resolution
models are available, joined by flexible linkers of unknown
structure. A simulated annealing optimization procedure is
employed to find the optimal positions and orientations of
the domains that are moved as rigid bodies attached to the
linkers, modeled as a chain of dummy residues separated
by 3.8 Å (Petoukhov et al. 2002). Starting from an arbitrary
conformation of domains and linkers, BUNCH performs
random modifications of the model maintaining the structures of the domains and the connectivity of the linkers. At
each step the scattering profile is computed and compared
with the experimental one, and in a simulated annealing
manner the model converges toward a configuration that
minimizes a merit function. The agreement with the
experimental scattering profile and some geometrical
parameters of the models are included in this merit function
(Petoukhov and Svergun 2005). The structural discrepancy
between the resulting models of 20 independent BUNCH
runs was evaluated with the program SUPCOMB (Kozin
and Svergun 2001) through the NSD parameter. NSD
values close to unity indicate that two models are similar,
and when this parameter departs from 1.0, the models
become more and more dissimilar.
Results
Initially, primary structures were compared to assess the
similarity among the L12/P proteins. Alignment of L12/P
protein sequences from all domains of life was attempted by
the authors of the previous analysis (Liao and Dennis 1994),
who found some similarities between them, but the
assumption of their common ancestry was less than certain.
However, we found no considerable similarity between
bacterial and archaeal/eukaryal L12/P proteins in our multiple sequence alignment of those proteins (Fig. 1). This
suggests that these two groups of proteins may not be evolutionarily related, or the relation, if any, could only be
preserved at a higher structural level. Therefore, the phylogenetic analysis was only applied to the archaeal and
eukaryal proteins. The phylogenetic trees of archaeal aL12
and eukaryal P proteins generated by the Bayesian, NJ, and
ME methods showed essentially the same topology (data not
show), and the tree obtained by the Bayesian inference is
shown in Fig. 2. The protein sequences were divided into
three well-supported clades, corresponding to P1, P2, and
aL12 proteins (Fig. 2). Within these three clades some
groups of sequences corresponded to known taxa, while
some of the other groupings did not reflect recognized
phylogenetic associations. Our results thus confirm earlier
123
123
----------MSITKDQ--IIEAVAAMSVMDVV--ELISAMEEKFGVSAAAAVAVAAGPVEAA---EEKTEFD-VILKAAGANKVAVIKAVRGATGLGLKEAKDLVESAPAALKEGVSKDDAEALKKALEEAGAEVEVK
----------MALTNED--IINAVSEMSVMQVV--ELIKAMEEKFGVTAAAATVAAAGPAAAAA--EEQTEFTIV-LAEAGDKKVNVIKVVRELTGLGLKEAKAVVDGAPGVVKEGASKEEAEAAKKALEEAGAKVELK
----------MAITKED--ILEAVGSLTVMELN--DLVKAFEEKFGVSAAAVAVAGPAGAGAADA-EEKTEFDVV-LASAGDQKVGVIKVVRAITGLGLKEAKDIVDGAPKTIKEGVSKAEAEDIQKQLEEAGAKVEIK
----------MALNIEE--IIASVKEATVLELN--DLVKAIEEEFGVTAAAPVAVAGGAAAGGAA-EEQSEFDLI-LAGAGSQKIKVIKVVREITGLGLKEAKELVDNTPKPLKEGIAKEEAEELKAKLEEVGASVEVK
----------MALNIEE--IIASVKEASVLELN--DLVKAIEEEFGVTAAAPVAVAAAGGAAA----EQTEFT-VELASAGDSKIKVIKVVREITGLGLKEAKELVDNAPKALKEGIAKDEAEEIKAKLEEVGANVEVK
----------MALNIEN--IIAEIKEASILELN--DLVKAIEEEFGVTAAAPVAVAAAGAGEAAAAKDS--FD-IELTAAGDKKVGVIKVVREITGLGLKEAKELVDGAPNVIKEGVAAAEAEELKAKLEEAGASVTLK
----------MALNIEN--IVAELENATILELS--ELVKAIEEKFDVTAAAPVAAAAGAGEAAAA-KDS--FD-VELTAAGDKKVAVIKEVRGITGLGLKEAKELVDGAPTVVKEGLSESEANEIKEKLEAAGASITLK
L12
L12
L12
L12
L12
L12
L12
Fig. 1 Alignment of representative ribosomal stalk L12/P proteins from three domains of life. Black boxes indicate fragments of sequences used for phylogenetic analysis, which also
correspond roughly to the most conserved N- and C-terminal domains (NTD and CTD) in eukaryotic proteins. The red boxes indicate the NTD and CTD in bacterial proteins
Escherichia coli
Pseudomonas aeruginosa
Neisseria meningitidis
Bacillus subtilis
Listeria monocytogenes
Streptococcus sanguinis
Lactococcus lactis
--------------MRYVASYLLAALGGNSSPSAKD-IKKILDSVGI-EADDDRLNKVISELNGK-NIEDVIAQGIGKLASVPA-GGAVAVSAAPGSAAPAAGSAPAAA------EEKKDEKKEESEESDDDMGFGLFD
--------------MRYVASYLLAALGGNSNPSAKD-IKKILDSVGI-EADDERLNKVISELNGK-NIEDVIAQGVGKLASVPA-GGAVAVSAAPGSAAPAAGSAPAAA------EEKKDEKKEESEESDDDMGFGLFD
--------------MRYVAAYLLAVLGGNESPTSKD-LKKILDSVGI-ETDDERLNKVISELNGK-NIEDVIAQGNGKLASMPA-GGAVAVSTGGVSAAPAAGAAPAAA------EEKKEEKKEESEESDDDMGFGLFD
--------------MRYVAAYLLAVLGGNTNPSAKD-IKNILGSVGI-EADDERLNKVVSELNGK-DINEVMNAGLSKLASVPA-GGAVAVSTASAGGGGGAPAEAPAA------EEKKEEKKEESEESDEDMGFGLFD
--------------MRYVAAYLLAVLGGKDSPANSD-LEKILSSVGV-EVDAERLTKVIKELAGK-SIDDLIKEGREKLSSMPV-GGGGAVAAADAAPAAAAGG--------DKKEAKKEEKKEESESEDDDMGFALFE
--------------MKFVAAYLLAVLAGNASPSAED-LTAILESVGC-EVDNERMELLLSQLSGK-DITELIAAGREKFASVPC-GGGGVAVAAASPAAGGAAPTA---------EAKKEEKVEEKEESDDDMGFSLFD
--------------MKVIAAYLLAVLGGNTSPTADD-VKSILESVGA-EADEEKLEFLLTELKDK-DITEVIAAGRERLSSVPS-GGGAIDMGAPAAVAGGGAAPA--------EEAKKEEKVEEKEESDEDMGFSLFD
--------------MKHLAAYLLLALAGNTSPSSED-VKAVLSSVGI-DADEERLNKLIAELEGK-DLQELIAEGSTKLASVPS-GGAAAAAPAAAGAAAGGAAAPAA----------EEKKEEEKEESDEDMGFGLFD
--------------MKYLAAYLLLVQGGNAAPSAAD-IKAVVESVGA-EVDEARINELLSSLEGKGSLEEIIAEGQKKFATVPT-GGASSAAAGAAGAAAGGDAA------------EEEKEEEAKEESDDDMGFGLFD
--------------MKYLAAYLLLNAAGN-TPDATK-IKAILESVGI-EIEDEKVSSVLSALEGK-SVDELITEGNEKLAAVPAA-GPASAGGAAAASGDAAA--------------EEEKEEEAAEESDDDMGFGLFD
--------------MQYLAAYALVALSGK-TPCKAD-VQAVLKAAGV-AIELSRVDALFQELEGK-SFDELMTEGRSKL--VGS--GSAAPAAAASTAGAAVAAAADA------------KKEASEEEADDDMGFGLFD
------------MAMKYVAAYLMCVLGGNENPSTKE-VKNVLGAVNA-DVEDEVLNNFIDSLKGK-SCHELITDGLKKLQNI--GGGVAAAPAGAAAVETAEA----------KKEDKKEEKKEEEEEEEDDLGFSLFG
--------------MRYVSAYLLAVLGGNANPKVDD-LKNILSAVGV-DADAETAKLVVSRLAGK-TVEELIAEGSAGLVSVS--GGAAPAAAAAPAAGGAAPAA----------DSKPAKKEEPKEESDDDMGFGLFD
----------MASVSELACIYSALILHDDEVTVTEDKINALIKAAGV-NVEPFWPGLFAKALANV-NIGSLICNV--------GAGGPAPAAGAAPAGGPAPSTAAAPA-----EEKKVEAKKEESEESDDDMGFGLFD
----------MASVSELACIYSALILHDDEVTVTEDKINALIKAAGV-NVEPFWPGLFAKALANV-NIGSLICNV--------GAGGPAPAAGAAPAGGPAPSAAAAPA-----EEKKVEAKKEESEESEDDMGFGLFD
----------MASVSELACIYSALILHDDEVTVTEDKINALIKAAGV-NVEPFWPGLFAKALANI-DIGSLICNV--------GAGGGAPAAAAPAGGAAPAGGGAAPA-----EEKKEEEKKEESEESDDDMGFGLFD
----------MASVSELACIYSALILHDDEVTVTEDKLNALIKAAGV-TIEPFWPGLFAKALASV-DIGSLICNV-------GAGGGAAPAAAAGAAAPAGGDAPA-------KEEEKKEEKKEESEESDDDMGFGLFD
----------MSTKAELACVYASLILVDDDVAVTGEKINTILKAANV-EVEPYWPGLFAKALEGI-NVKDLITNI-------GSGVGAAPAGGAAPAAAAAAPAA--------ESKKEEKKKEEESDQSDDDMGFGLFD
----------MASNQELACVYAALILQDDEVAITGEKIATLLKAANV-EFEPYWPGLFAKALEGV-DVKNLITSVSS-------GAGSGPAPAAAAAAPAAGGAAPAA---------ETKKKEEPKEESDDDMGFGLFD
-----------MASGELACRYAALILSDDGIAITAEKIATIVKAANI-KVESYWPALFAKLLEKR-NVEDLILSV-------GSGGGAAPVAAAAPAGGAAAAAAPAV----------EEKKEEAKEESDDDMGFSLFD
-----------MSTAELACSYAALILADDGVEITADKIQTLLGAAKVADVEPIWTSLFAKALEGK-DIKDLLTNV-------GSGGAAAPAAVGGAAAGAAAPAEAAAA---------EEKKEEEKEESDEDMGFGLFD
MASIPASELPECEKQELLCTYAALILHEEKMSITSDNILKLIKNSNN-TVLPYLPMLFERALKGK-DIQSLLSNLSV-----GSAPAAAAQVTT--------------EKPSEDKKEAKKEEKVEEEEEEDDLGFSLFG
------------MSTESALSYAALILADSEIEISSEKLLTLTNAANV-PVENIWADIFAKALDGQ-NLKDLLVN--------FSAGAAAPAGVAGGVAGGEAGEA-----------EAEKEEEEAKEESDDDMGFGLFD
-------------MSDSIISFAAFILADAGLEITSDNLLTITKAAGA-NVDNVWADVYAKALEGK-DLKEILSG----------FHNAGPVAGAGAASGAAAAGGDAAA--------EEEKEEEAAEESDDDMGFGLFD
-----------MTTETLACTYAALMLSDAGLPTSAENIAAAVKAAGV-SVRPTMPIIFARFLEKK-SVEALMAA-------AATQAPTATSAAAAPAAGEASGKA------------EEKKKEEPEEEGDDDMGFGLFD
--------------MEY--VYAALLLHSVGKEINEENLKAVLQAAGV-EPDEARIKALVAALEGV-NIDEVIEK--------AAMPVAVAAAPAAAPAGGG------EEKKEEEKKEEEEKEEEVSEEEALAGLSALFG
--------------MEY--VYAALLLHAAGKEITEENLKAVLEAAGV-TPDEARIKALVAALEGV-NIDEVIEK--------AAMPVAAPVAVAAAPAAEGGAAEAAQ-------EEEEEEEEEASEEEALAGLGALFG
--------------MEY--IYASLLLHAAKKEISEENIKNVLSAAGI-TVDEVRLKAVAAALKEV-NIDEILKT-------ATAMPVAAVAAPAGQQTQQAA---------EKKEEKKEEEKKGPSEEEIGGGLSSLFG
--------------MEY--VYGALLLHAAGKEVDEEKLKKVLEDVGV-QVDEARLKTLVSGLKDV-NIDEVLKN-------ASVAQVATAPAPAA--------EEKKKEEPKKKEEKKKEEDKEHEEEEAMSGLSALFG
--------------MEY--VYGALLLHAAGKEVDEEKLKKVLEDVGV-QVDEARLKTLVSGLKDV-NIDEVLKN--------ATVAPVAAAPAAPQE---------KKEEPKKKEEKKKEENKEQEEEEAMSGLSALFG
--------------MEY--IYAALTLNESGESITEDAVTEVLEAAGV-DVEDSRVKALVAALEDV-DIEEAIDT----AAAAPAPAAGAGAAS---EAEVEDEADTDEEEPDETEEEEADEADDEDDEASGEGLGNLFG
--------------MEY--VYAALILHESGEELNEDNLTDVLDAAGV-DVEESRVKALVAALEDV-DVEEAIET----AAAVPAGGAGGAAAGGAA-EADEAEEADEGGDADEAEEAEEEAEDDGDDEDGGEGLGELFG
--------------MEY--IYAALLLHNAGKDITEESVSAVLSAAGT-EVNESRAKALVAALEDV-DIEEAMAT--------AAFAPAAAVVAAPVAETAA-------------EEVPAEENKAEEEESGMAGLGALFG
CTD
P2 Homo sapiens
P2 Rattus norvegicus
P2 Gallus gallus
P2 Danio rerio
P2 Drosophila melanogaster
P2A Zea mays
P2B Zea mays
P2 Aspergillus fumigatus
P2B Saccharomyces cerevisiae
P2A Saccharomyces cerevisiae
P2 Leishmania brasilensis
P2 Plasmodium falciparum
P2 Caenorhabditis elegans
P1 Homo sapiens
P1 Rattus norvegicus
P1 Gallus gallus
P1 Danio rerio
P1 Drosophila melanogaster
P1 Caenorhabditis elegans
P1 Zea mays
P1 Aspergillus fumigatus
P1 Plasmodium falciparum
P1A Saccharomyces cerevisiae
P1B Saccharomyces cerevisiae
P1 Leishmania peruviana
aL12 Pyrococcus furiosum
aL12 Thermococcus kodakarensis
aL12 Susfolobus solfataricus
aL12 Thermoplasma acidophilium
aL12 Thermoplasma volcanium
aL12 Haloquadratum walsbyi
aL12 Natromonas pharaonis
aL12 Methanococcoides burtonii
NTD
10
20
30
40
50
60
70
80
90
100
110
120
130
140
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|.....
158
J Mol Evol (2008) 67:154–167
159
aL12 Sulfolobus solfataricus
a ii
tokod
sis
lobus
m
ren
Sulfo
hilu
ka
idop
da
ko
a ac
cus
cco
op
erm
aL12
2Th
aL1
aL
lasm
is
12
on
ra
as
o
erm
Th
aL
12
Py
on
m
no
co
io
su
0.82/0.73/-
0.53/82
0.90/75
1.0/98
A
R
lte
rn
ar
P2
ia
Ho
al
mo
te
P2 G
s ap
r
allus
ien 0.98/95 na
t
gallu s
0.96/91 a
s
P2 Danio rerio
0.92/63
sn
tu
at
g
ve
or
0.59/-
0.69/0.57/84
0.54/-
/54
1.0/-
0.99/82
sis
dop
abi
Ar
P2
cc
ha
substitutions/site
m
iu
tel
os
y
ict
P1
Ze
s
tu
lla
ici
P1
D
n
pe
ce
s
m
ro
yc
es
ae
isi
rev
ce
ce
0.1
a
am
lis
ica
rop
his
s t gicus
rc
u
o
p
0.99/74
ly
no rve
Po
Xe s no
0.89/0.77/P1
ns
P1 attu
0.83/sapie
R
2
/9
1
0.94/PP1 Homo
0.67/- 1.0
0.54/0.83 0.58/- P P1 Ga
1D
/- 0
ll
1.0/66
0.59/.71/6
ani us gallus
6
o re
0.75/rio
1.0/P
P
P
1
1.0/99 1 D 1 B Art
0.99/ro omb emia sa
lina
yx
so
0.97/ph
mo
0.50/ri
ila
m
ela
1.0/no
ga
ste
1.0/r
0.99
1.0/88
P2
B
tha Zea
lian m
a ay
s
P2A
Zea
P2 S
may
chizo
s
sacch
arom
yces p
ombe
P2B Sacc
haromyc
es cerevi
siae
P2A Saccharo
myces cerevisia
e
P2A T
rypan
osoma
cruzi
P2 Leishmania brasiliensis
ma cruzi
P2A Trypanoso
0.98/7/-0.83/0.9
/
1.0
Sa
la
P
P2
s
ovi
a
iv
ia b m
bes aru sat
Ba
p
i
2
za
c
P
l
y
a
r
f
O
ium
P2
od
sm
ys
0.93/66
m
eu
oid
sc
di
s
an
leg
ga
se
ha
iti
op
bd
ves
ha s bre
or
u
en hei
us
c
t
Ca 1 Os
iga
P1 P
um
sf
sa
ras
illu
rg
ra c
pe
spo
As euro
ternata
P1 P1 N
naria al
P1 Alter
P1 Clad
osporiu
m herba
rum
P1A
Sch
izos
acc
h
P1 Pl
a
rom
P1
asmod
yces
A
ium f
P1
Sa
pom
alc
N
i
p
c
a
e
r
be
c
o
u
m
sp o
ha
ra c
ro
anin
my
um
P2 Artemia salina
0.61/aster
lanog ns
ori 0.86/a
ila me
osoph
eleg byx m1.0/r
s
D
i
2
t
i
P
m
abd
Bo
orh
P2 0.97/aen
P2 C
Na
1.0/86
1.0/59
m
B
P2
herbarumatus sa
sporium
ig
as
P2 Clado
s fum ra cr
gillu
spo
sper
uro
P2 A
Ne
P2
us
ic
sf
ur
2
L1
a
cu
P2
tro
1.0/89
ro
c
a
ph
P1
Fig. 2 Phylogenetic tree of
aL12/P proteins obtained by
Bayesian inference. Numbers at
nodes represent Bayesian
posterior probabilities/NJ
bootstrap support of particular
clades. (–) That clade does not
exist in NJ analysis
aL12
Meth
anoth
ermo
aL1
bacte
2M
r ther
mauto
eth
a
troph
noc
aL
icus
o
12
cco
ide
Ha
s
loq
bur
P1
ton
ua
Try
ii
dr
P1
pan
atu
oso
Le
m
m
i
a
s
P1
hm
wa
cru
Ch
zi
lsb
an
lam
ia
yi
pe
yd
ru
om
P1 P1 A
v
o
ian
P1
O rab
na
ry
i
sr
Tet
a
za dops
rah
is t einha
sa
ym
hal
tiv
ena
ian rdtii
a
the
a
mo
ph
ila
J Mol Evol (2008) 67:154–167
v
re
isi
ae
P1 Homo sapiens
Jpred
----HHHHHHHHHHHHH----HHHHHHHHHHHHHH------HHHHHHHHHHH-----HHHHH------------HHHHHHHHHHHHH-HHHHHHHHHH---------------Sspro
---HHHHHHHHHHHHH----HHH-HHHHHHHHHH---------HHHHHHHHH----HHHHHE--------------------------HHHHHHH-HH---------------P1A Saccharomyces cerevisiae
Jpred
--HHHHHHHHHHHHH----HHHHHHHHHHHHHH-------HHHHHHHHHH-----HHHH--------HHHHHHHHHHHHHHHHHHHHHHH----------------Sspro
--HHHHHHHHHHHHH----HH-HHHHHHHHHH-------HHHHHHHHHH----HHHHHHE---------------------HHHHHHHHHHHHHH-----------P2 Homo sapiens
Jpred
Sspro
P2B Saccharomyces cerevisiae
Jpred
Sspro
aL12 Susfolobus solfataricus
Jpred
Sspro
Escherichia coli
L7/L12
--HHHHHHHHHH-------HHHHHHHHHH----HHHHHHHHHHHHH----HHHHHHHH-------------------HHHHHHHHHHHHHH--HHHHH----------------HHHHHHHHHHH-------HHHHHHHHHH------HHHHHHHHHHH----HHHHHHH-HHHH-------------------------HHHHHHH-H-------------------HHHHHHHHHH-------HHHHHHHHHH----HHHHHHHHHHHHH-----HHHHHHHH---------------HHHHHHHHHHHHHHHHHHH----------------HHHHHHHHHH--------HHHHHHHHHH------HHHHHHHHHHH-----HHHHHHH-------------HHHHHHHHHHH----HHHHHHHHHHHHH-----------HHHHHHHHHH--HHHHHHHHHHHHHH----HHHHHHHHHHHHHHH--HHHHHHHH---HHHHHH----HHHHHHHHHHHHH-----------------------HHHHHHHHHHHHHHHH-HHHHHHHHHH----HHHHHHHHHHHHHHHH-HHHHHHHH----HHHHH----HHHHHHHHHHHHHHHHH---------------------HHHHHHHHH----HHHHHHHHHHHHH-----------------------EEEEEEEEE----HHHHHHHHHHH---HHHHHHHH---EEEEEEEHHHHHHHHHHHHHHH----EEEEE
Fig. 3 Secondary structure of the whole proteins as determined by the Jpred and Sspro programs. The secondary structure of L7/L12 was
extracted from known 3D structures (PDB nos: 1RQT, 1RQS). H, a-helical structure; E, b-sheet
findings of Liao and Dennis (1994), but only with respect to
relationships between archaeal and eukaryal proteins.
Since evolutionary relationships are not always well
recognized at the level of primary structure, we performed
a set of structural analyses to evaluate the secondary and
tertiary structure of the L12/P proteins. First, a secondary
structure analysis was performed using three representative
proteins, the H. sapiens, S. cerevisiae and S. solfataricus,
with the aid of the Jpred and Sspro programs. The
secondary structure (2D) of E. coli L7/L12 protein was
extracted from known 3D models (Bocharov et al. 2004;
Leijonmarck et al. 1980). Once again, the aL12/P proteins
could be easily aligned, with the 2D organization of aL12
closely resembling that found in the P-protein group
(Fig. 3). The bacterial L7/L12 did not show significant
similarities in the 2D structure to the archaeal/eukaryal
aL12/P proteins, except for the first 30 amino acids, which
form two a-helices responsible for dimerization.
123
160
J Mol Evol (2008) 67:154–167
Fig. 4 Experimental X-ray
scattering patterns of L12/P
proteins and scattering
computed from their models.
Dots with error bars depict
experimental data, and solid
lines are fits obtained from the
ab initio models. The insets
show the distance distribution
functions [p(r)] computed from
experimental data by GNOM.
(A, B) Human P1-P2 and P2-P2
dimers, respectively; (C) aL12
archaeal S. solfataricus dimer;
(D) L7/L12 bacterial E. coli
dimer
Table 1 Structural parameters of L12/P proteins computed from scattering data
Sample/parameter
Rg (nm)
Dmax (nm)
MM (kDa)
MMseq (kDa)
v
Human P1-P2
3.64 ± 0.07
12.0 ± 0.5
29 ± 4
23.2
1.13
Human P2-P2
3.25 ± 0.05
11.0 ± 0.5
24 ± 3
23.3
1.06
aL12
3.35 ± 0.07
12.0 ± 0.5
23 ± 2
23.8
1.05
L7/L12
3.58 ± 0.06
12.0 ± 0.5
21 ± 2
22.8
0.95
Note: Rg, Dmax, and MM are radii of gyration, maximum sizes, and molecular masses, respectively, calculated from the scattering data. MMseq
are molecular masses of the solutes predicted from the sequences, and v is the discrepancy between the experimental data and the computed
scattering from the most probable GASBOR models
123
J Mol Evol (2008) 67:154–167
Fig. 5 Ab initio models of the
L12/P proteins. P1-P2 and P2P2, human hetero- and
homodimers, respectively; the
complexes were resolved at 1.9nm resolution. P1A-P2B, S.
cerevisiae dimer (Grela et al.
2007); resolution, 0.7 nm. aL12,
S. solfataricus dimer;
resolution, 1.8 nm. L7/L12 E.
coli dimer; resolution, 1.8. All
models are shown in the front
view (A) and the view rotated
by 90° around the Y-axis (B).
The models were generated with
the VMD program (Humphrey
et al. 1996)
161
y
A
B
x
P1-P2
P2-P2
P1A-P2B
aL12
L7/L12
To provide a more exhaustive insight into the issue,
tertiary structures of proteins were analyzed with the aid of
SAXS. The structures were solved for human P1-P2 and
P2-P2 dimers, S. solfataricus aL12 dimer, and E. coli L7/
L12 complex. The experimental scattering pattern of all
complexes is presented in Fig. 4, and the structural
parameters computed from these data are listed in Table 1.
A low-resolution SAXS model of the human P1-P2
heterodimer was reconstructed ab initio as described under
Materials and Methods. Twelve independent reconstructions using the program GASBOR yielded superimposable
results providing reasonable fits to the experimental data.
The ab initio model computed by the program DAMAVER
is presented in Fig. 5. The model displays an elongated
123
162
bent shape consisting of two domains with a maximum
diameter of about 12 nm and a cross section of about 3 nm
(Fig. 4A). However, the estimated molecular mass (MM)
is somewhat larger than the value expected from the
sequence, which indicates that a fraction of the protein
may undergo aggregation in solution. This information is
supported by our observation that the human heterocomplex has a tendency to aggregate (Grela et al. 2008). But
there was no significant increase in the apparent MM of the
solute at higher protein concentrations, which should have
been the case if unspecific protein aggregation or extensive
formation of oligomers were taking place. Instead, the
Guinier plots of the experimental data displayed a linear
behavior characteristic of monodisperse solutions. It could
thus be concluded that the protein is largely dimeric in
solution, however, a small portion of the dimer may exist
in higher oligomeric forms which may slightly increase the
experimental MM value. Additionally, since the human
P2-P2 homodimer may exist in solution as a stable entity
and may represent a transient form during the exchangeability of the P proteins (Grela et al. 2008), we used SAXS
to determine the overall shape of the homodimer as well.
Low-resolution shape reconstructions of the homodimer,
performed assuming P2 symmetry and without symmetry
restrictions, lead to superimposable shapes. The distance
distribution function of the homodimer (Fig. 4B, inset)
indicates an elongated particle with a maximum diameter
of 11 nm and a cross section of about 2.5 nm. A shoulder
at higher intraparticle distances observed in the p(r)
function suggests that the average separation between the
centers of two domains is about 6 nm. The ab initio model
is displayed in Fig. 5. The shape of the homodimer clearly
displays two linearly positioned domains with a waist in
the middle. In the case of human P2-P2 dimer the estimated MM of the solute agrees very well with the value
expected from the sequence, indicating that the protein is
monodisperse in solution. As the next step, we analyzed
the structure for the orthologous archaeal protein aL12.
The structural parameters are almost identical to those
obtained for the eukaryotic counterparts (Table 1). Analysis of the aL12 protein showed a good agreement between
theoretical and estimated MM, underscoring the fact that
the protein exists as a single species in solution, which was
also verified by size exclusion chromatography and native
PAGE analyses (data not shown). The distance distribution
function of the aL12 dimer (Fig. 4C, inset) indicates an
elongated particle with a maximum diameter of 12 nm and
a cross section of about 2.5 nm. Also in this case, a
shoulder at higher intraparticle distances was observed in
the p(r) function, suggesting that the average separation
between the centers of two domains is about 6 nm. Following the procedure presented above, an ab initio model
was constructed (Fig. 5). The shape of the aL12 dimer
123
J Mol Evol (2008) 67:154–167
Fig. 6 Rigid-body modeling of the L7/L12 protein dimer. (A)
Experimental X-ray scattering pattern of the L7/L12 dimer. Dots
with error bars depict experimental data; the red line represents the fit
from the rigid-body model. (B) Rigid-body models of the L7/
L12 protein. The N-terminal domain and C-terminal domain are
shown in green and blue, respectively; the linker is marked in red.
Models are shown in the top view (I) and front view (II), rotated by
90° around the X-axis. (C) Ab initio SAXS model of L7/L12 proteins
(front view). The models were generated by the VMD program
J Mol Evol (2008) 67:154–167
clearly resembles the structure of the eukaryotic complexes, with two linearly positioned domains with a waist
in the middle.
There is no available SAXS model for bacterial L7/L12
protein, and available structural data for the bacterial dimer
are not suitable for direct comparison with the archaeal/
eukaryal SAXS models. Therefore, in order to obtain
compatible structural model of bacterial proteins, the
SAXS analysis was performed with the E. coli L7/L12
dimer. The structure of the individual domains for that
protein has been solved by crystallography (Leijonmarck
et al. 1980; Wahl et al. 2000) and NMR spectroscopy
(Bocharov et al. 2004), but the overall shape of the whole
dimer in solution or of the stalk has not been solved, except
for a provisional proposition presented recently (Diaconu
et al. 2005). Consequently, we were able to obtain the
ab initio low-resolution model of L7/L12, as depicted in
Fig. 5. Analysis of the E. coli L7/L12 dimer showed that
the protein exists in solution as a monodisperse entity, even
at very high protein concentrations, and the estimated MM
of the solute agrees well with the value computed from the
L7/L12 sequence (Table 1). The p(r) function (Fig. 4D,
inset) indicates an elongated particle with the maximum
diameter of 12 nm.
The L7/L12 dimer displays three distinct and well-separated domains. The two C-terminal domainss (CTDs) are
very well recognizable and one centrally located N-terminal domain (NTD) can also be identified confidently. The
angle between the two CTDs is of about 120o between the
directions of their long axes. It should be emphasized that
this is the first insight into the molecular organization of
individual domains of the L7/L12 dimer under native
conditions and it fully agrees with the propositions made
previously (Bocharov et al. 2004; Mulder et al. 2004).
Moreover, to support our L7/L12 model, the experimental
scattering profile was fitted with the rigid-body modeling
program BUNCH in order to find the spatial arrangement
of the available high-resolution structures representing the
folded domains (see Materials and Methods). The program
was run 20 times from random starting configurations, and
an excellent agreement with the SAXS curve was obtained
in all cases, with v values between 1.04 and 1.33, as shown
in Fig. 6A. The average NSD obtained between the 20
structural models derived was 0.88 ± 0.07, indicating that
all structures present an almost-equivalent domain distribution. Derived structural models present an open arm
shape, where the NTD is located at the elbow and the two
CTDs are at the extremes. The average interdomain distances between both CTDs and between the NTD and the
CTDs are 85.0 ± 2.2 and 48.2 ± 0.4 Å, respectively, and
the angle formed between the three domains is 124.0 ±
5.8°. Therefore, the measured SAXS profile tightly constrains the relative domain positions in L7/L12, although
163
their orientations and linker conformations varied from
model to model. This result produced a remarkable fit, at
the level of resolution available between SAXS and
BUNCH models, and fully confirmed the domain orientation in the SAXS model (Figs. 6B and C).
Summing up, the obtained archaeal/eukaryal SAXS
models, which can be described as two linearly positioned
domains with a waist in the middle, depart significantly
from the bacterial one, which showed a typical threedomain organization, and moreover, this structure suggests
the presence of limited amount of dynamics in the linkers
connecting the NTD and the CTD in L7/L12. Therefore,
considering 1D, 2D, and 3D analyses, there is no similarity
between bacterial and archaeal/eukaryal proteins.
Discussion
Phylogenetic studies of ribosomal RNA have revolutionized our understanding of biological diversity by revealing
that modern organisms fall into three phylogenetic
domains: Bacteria, Archaea, and Eukarya (Woese et al.
1990). In principle, the rRNA sequence is well suited for
determining deep phylogenetic relationships because rRNA
is present in all organisms, and has evolved at a sufficiently
slow rate to retain phylogenetic information between distantly related organisms. On the other hand, phylogenetic
analysis of amino acid sequences does not always determine clear evolutionary relationships among the proteins of
interest. Systematic phylogenetic analyses of universally
conserved proteins have revealed a small set of genes that
can be traced back to the universal ancestor and have
coevolved since that time with the ribosomal RNA since
their divergence from a last universal common ancestor
(LUCA). As found, most of the core three-domain genes/
proteins belong to the nucleic acid-based central information pathway (ribosomal proteins, DNA/RNA polymerase
subunits, ribosomal elongation factors) and more than half
are ribosomal proteins (29 of 50) (Harris et al. 2003),
supporting the conclusion that the divergence of the three
types of ribosomes (bacterial, archaeal, and eukaryal)
occurred after a relatively efficient ribosome structure had
already been formed (Olsen and Woese 1997). The
majority of the universally conserved ribosomal proteins
showing a three-domain phylogeny is represented by smallsubunit proteins. The proteins of the large ribosomal subunit are a more complex group, and a smaller fraction of
these was universally conserved (Harris et al. 2003). In
total, 32 ribosomal proteins are strictly conserved in all
bacterial, archaeal, and eukaryotic ribosomes which ensure
preservation of the core and global shape of the ribosome
(Lecompte et al. 2002). The majority of orthologous ribosomal proteins show a clear sequence-based relationship
123
164
reflected by functional link. However, the proteins that
form the ribosomal stalk do not fully obey this rule. The
base of the prokaryotic/eukaryotic stalk is formed by a very
conserved protein, L10/P0, which shares the ‘‘threedomain’’ phylogenetic rRNA topology (Liao and Dennis
1994). Having such properties, these proteins have also
been classified as universally conserved genes, belonging
at the same time to the genetic core that traces its ancestry
back to the LUCA of life (Harris et al. 2003). While
the evolutionary connection between L10 and P0 from
the three domains is unambiguous, the relationships among
the L12/P proteins are less obvious. Several evolutionary
analyses have linked those proteins, implying that they
might have a common evolutionary origin (Liao and
Dennis 1994; Ramirez et al. 1989), however, the path of
evolution is still obscure. There is no doubt that the L12/P
proteins play identical roles on the ribosome, being part of
the GTPase-associated center which is directly responsible
for the stimulation of translation-factor-dependent GTP
hydrolysis (Rodnina et al. 2000). However, our present
analyses, considering all structural levels, provide evidence
that these proteins have different structural organization
and in fact may not be orthologous.
In our initial analyses, inspection of a multiple sequence
alignment did not indicate a close relationship between the
bacterial and the archaeal/eukaryal L12/P proteins, implying
that they may represent two unrelated sequence types. Phylogenetic analysis confirms close relationships between
archaeal and eukaryal proteins. The placement of the root of
the tree of life is still an open question (Gribaldo and Philippe
2002; Philippe and Forterre 1999; Walsh and Doolittle
2005). Even though we follow herein the view on the original
position of the LUCA (Woese 1998; Woese et al. 1990), it
must be pointed out that the postulated replacement event on
the ribosomal stalk has taken place on the branch between
Bacteria and Archaea plus Eukarya, irrespective of the actual
position of the root. In fact, the structural similarity of L12/P
ribosomal proteins in Archaea and Eukarya is yet another
synapomorphy, linking the two groups together.
A secondary structure comparison confirmed this conjecture by showing that there are significant differences
between the analyzed proteins. The NTD of L7/L12, which
is responsible for dimerization, is composed of 30 amino
acids and has two a-helices (Bocharov et al. 2004). On the
other hand, the NTD of aL12/P proteins, which is also
responsible for protein-protein interactions, contains about
65 amino acids and has four a-helices (Jose et al. 1995;
Tchorzewski et al. 2003). At first glance, one may find
some resemblance in the 2D structural organization, but it
should be noted that these two domains are of profoundly
different sizes. Even more pronounced differences are seen
in the C-terminal parts. The C-terminal segment of L7/L12
proteins, consisting of 68 amino acids (residues 53–120), is
123
J Mol Evol (2008) 67:154–167
considered a functional element and adopts a compact
globular a/b-fold (Leijonmarck et al. 1980). On the other
hand, the CTD of the L12/P proteins is also regarded as a
functional part (Santos and Ballesta 1995) and, in principle,
should be conserved throughout evolution, but that is not
the case here. Our prediction indicates that the C-terminal
fragment of the P proteins, comprising only some 20 residues, does not form a compact domain, although the first
half of this region has a propensity to form an a-helix and
the second half folds into a turn-like conformation at low
temperatures, as shown before (Soares et al. 2004).
Therefore, it seems that these two groups of proteins have a
totally different 2D organization in the C-terminal part,
which in turn may reflect a very distinct 3D organization.
The results of the 2D analysis are fully supported by the
structural study using SAXS, which provided convincing
evidence that the bacterial and archaeal/eukaryal L12/P
proteins are not structurally related. The archaeal/eukaryal
aL12/P proteins exhibit a very similar spatial organization.
Both aL12 and P dimers have a very elongated, slightly
dumbbell-shaped structure with a typical waist separation. In
this structure the CTDs are present as separate domains, but
as we have already suggested the movement of the C-terminal segments relative to the central NTD dimer makes
them appear to have wider cross sections than the central
portions (Grela et al. 2007). On the other hand, the SAXS
model for the bacterial L7/L12 dimer showed a structural
organization entirely different from that of their archaeal/
eukaryal counterparts. In the dimer, the three structural
domains are very well recognized, with the NTD located
centrally and the two CTDs placed at the two termini, which
is in excellent agreement with the available 3D structures
(Bocharov et al. 2004) and also as shown by rigid-body
modeling. The hinge region requires special attention; in this
model the two hinges have unequal lengths, which might
suggest that one is structured, probably adopting an a-helical
form, and the second one is in a relaxed conformation. This
observation may provide support for models presented previously, where it was suggested that the hinge regions of the
L7/L12 protein dimer might undergo an a-helix coil transition in aqueous solution, whereas in a nonpolar environment,
it prefers to fold into long a-helices, which would close the
hydrophobic cleft of the NTD (Bocharov et al. 2004;
Chandra Sanyal and Liljas 2000; Wahl et al. 2000). It should
be emphasized that this is the first insight into the spatial
organization of individual NTDs and CTDs of the whole L7/
L12 complex in solution. All these experiments suggest that
the bacterial and archaeal/eukaryal L12/P proteins belong to
different groups that are not structurally related. This
observation is in line with the earlier data showing that they
are not interchangeable between Bacteria and Archaea/
Eukaryotes, despite their identical functions (Uchiumi et al.
1999).
J Mol Evol (2008) 67:154–167
It seems that a rudimentary GTPase-associated center
was present in the LUCA in the form of L10/P0 protein and
some additional rRNA elements such as a sarsin-ricin loop,
which could support the basic activity of this center and
probably enabled the transition from progenotes to genotes,
but then much of the stalk structure evolved in a very
dynamic way in the bacterial and the archaeal/eukaryotic
lines of descent, with a significant further refinement
occurring in the eukaryotic line once it separated from the
Archaea, as was the case for other ribosomal processes
(Kyrpides and Woese 1998). On the basis of our results the
two main evolutionary paths could be considered for L12/P
proteins. The first path, convergent evolution, represents, in
our view, the most likely scenario, where the bacterial and
the archaeal/eukaryal L12/P proteins probably appeared on
the ribosomal particle as two independent events in the
evolution of the GTPase center, to fulfill the special needs
for further adjustment of speed and accuracy of the ribosomal machinery and, also, as has been postulated
(Krokowski et al. 2007; Remacha et al. 1995), to bring a
regulatory aspect into protein synthesis. The second path,
where the L12/P proteins come from an ancestral protein in
the LUCA genome, but would have diverged to the point
that no similarity can be detected, cannot be ruled out.
However, the first scenario seems more likely. First, the
structural differences on all levels of protein structure are
so dramatic that most likely they are related to two
autonomous evolutionary incidents leading to independent
creation of two protein structures with the same function.
Second, the ribosomes deprived of those proteins have
residual activity toward stimulation of translation factordependent GTP hydrolysis (Mohr et al. 2002), which may
indicate that addition of the stalk proteins was a relatively
late event, realized after all basic elements of the GTPaseassociated center in the LUCA was put in place. Third, the
P0 stalk protein is homologous to L10 and they show
sequence similarity (Maki et al. 2007). If L12 has been
coevolving with the P1/P2 protein, it should have been
showing the same pattern of sequence and structural
change. Consequently, we postulate that these proteins
should be regarded as analogous rather than homologous.
Acknowledgments This work was supported by the Ministry of Science and Higher Education grant 2 P04A 004 29, 2005/2008. The SAXS
measurements were supported by the European Community-Research
Infrastructure Action under the FP6 ‘‘Structuring the European Research
Area Programme,’’ contract number RII3/CT/2004/5060008. P.B. was
supported by funds from the Ramóny Cajal research program.
References
Ballesta JP, Remacha M (1996) The large ribosomal subunit stalk as a
regulatory element of the eukaryotic translational machinery.
Prog Nucleic Acid Res Mol Biol 55:157–193
165
Bocharov EV, Sobol AG, Pavlov KV, Korzhnev DM, Jaravine VA,
Gudkov AT, Arseniev AS (2004) From structure and dynamics
of protein L7/L12 to molecular switching in ribosome. J Biol
Chem 279:17697–17706
Boulin CJ, Kempf R, Gabriel A, Koch MHJ (1988) Data acquisition
systems for linear and area X-ray detectors using delay line
readout. Nucl Instrum Meth 269:312–320
Chandra Sanyal S, Liljas A (2000) The end of the beginning:
structural studies of ribosomal proteins. Curr Opin Struct Biol
10:633–636
Cheng J, Randall AZ, Sweredoski MJ, Baldi P (2005) SCRATCH: a
protein structure and structural feature prediction server. Nucleic
Acids Res 33:W72–W76
Cuff JA, Clamp ME, Siddiqui AS, Finlay M, Barton GJ (1998) JPred:
a consensus secondary structure prediction server. Bioinformatics 14:892–893
Diaconu M, Kothe U, Schlunzen F, Fischer N, Harms JM, Tonevitsky
AG, Stark H, Rodnina MV, Wahl MC (2005) Structural basis for
the function of the ribosomal L7/12 stalk in factor binding and
GTPase activation. Cell 121:991–1004
Feigin LA, Svergun DI (1987) Structure analysis by small-angle
X-ray and neutron scattering. Plenum Press, New York
Frank J, Gao H, Sengupta J, Gao N, Taylor DJ (2007) The process of
mRNA–tRNA translocation. Proc Natl Acad Sci USA
104:19671–19678
Gabriel A, Dauvergne F (1982) The localization method used at
EMBL. Nucl Instrum Method 201:223–224
Gonzalo P, Reboud JP (2003) The puzzling lateral flexible stalk of the
ribosome. Biol Cell 95:179–193
Gonzalo P, Lavergne JP, Reboud JP (2001) Pivotal role of the P1
N-terminal domain in the assembly of the mammalian ribosomal
stalk and in the proteosynthetic activity. J Biol Chem
276:19762–19769
Grela P, Helgstrand M, Krokowski D, Boguszewska A, Svergun D,
Liljas A, Bernado P, Grankowski N, Akke M, Tchorzewski M
(2007) Structural characterization of the ribosomal P1A-P2B
protein dimer by small-angle X-ray scattering and NMR
spectroscopy. Biochemistry 46:1988–1998
Grela P, Sawa-Makarska J, Gordiyenko Y, Robinson CV, Grankowski
N, Tchorzewski M (2008) Structural properties of the human
acidic ribosomal p proteins forming the p1-p2 heterocomplex.
J Biochem 143:169–177
Griaznova O, Traut RR (2000) Deletion of C-terminal residues of
Escherichia coli ribosomal protein L10 causes the loss of
binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer
are active. Biochemistry 39:4075–4081
Gribaldo S, Philippe H (2002) Ancient phylogenetic relationships.
Theor Popul Biol 61:391–408
Guarinos E, Remacha M, Ballesta JP (2001) Asymmetric interactions
between the acidic P1 and P2 proteins in the Saccharomyces
cerevisiae ribosomal stalk. J Biol Chem 276:32474–32479
Guinier A (1939) La diffraction des rayons X aux tres petits angles;
application a l’etude de phenomenes ultramicroscopiques. Ann
Phys (Paris) 12:161–237
Harris JK, Kelley ST, Spiegelman GB, Pace NR (2003) The genetic
core of the universal ancestor. Genome Res 13:407–412
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference
of phylogenetic trees. Bioinformatics 17:754–755
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular
dynamics. J Mol Graph 14:33-8, 27–28
Hury J, Nagaswamy U, Larios-Sanz M, Fox GE (2006) Ribosome
origins: the relative age of 23S rRNA Domains. Orig Life Evol
Biosph 36:421–429
Ilag LL, Videler H, McKay AR, Sobott F, Fucini P, Nierhaus KH,
Robinson CV (2005) Heptameric (L12)6/L10 rather than
canonical pentameric complexes are found by tandem MS of
123
166
intact ribosomes from thermophilic bacteria. Proc Natl Acad Sci
USA 102:8192–8197
Jose MP, Santana-Roman H, Remacha M, Ballesta JP, Zinker S
(1995) Eukaryotic acidic phosphoproteins interact with the
ribosome through their amino-terminal domain. Biochemistry
34:7941–7948
Koch MHJ, Bordas J (1983) X-ray diffraction and scattering on
disordered systems using synchrotron radiation. Nucl Instrum
Meth 208:461–469
Konarev PV, Volkov VV, Sokolova AV, Koch MHJ, Svergun DI
(2003) PRIMUS—a Windows PC-based system for small-angle
scattering data analysis. J Appl Crystallogr 36:1277–1282
Kozin MB, Svergun DI (2001) Automated matching of high- and lowresolution structural models. J Appl Crystallogr 34:33–41
Krokowski D, Boguszewska A, Abramczyk D, Liljas A, Tchorzewski M,
Grankowski N (2006) Yeast ribosomal P0 protein has two separate
binding sites for P1/P2 proteins. Mol Microbiol 60:386–400
Krokowski D, Tchorzewski M, Boguszewska A, McKay AR, Maslen
SL, Robinson CV, Grankowski N (2007) Elevated copy number
of L-A virus in yeast mutant strains defective in ribosomal stalk.
Biochem Biophys Res Commun 355:575–580
Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5:150–163
Kyrpides NC, Woese CR (1998) Archaeal translation initiation
revisited: the initiation factor 2 and eukaryotic initiation factor
2B alpha-beta-delta subunit families. Proc Natl Acad Sci USA
95:3726–3730
Lecompte O, Ripp R, Thierry JC, Moras D, Poch O (2002)
Comparative analysis of ribosomal proteins in complete
genomes: an example of reductive evolution at the domain
scale. Nucleic Acids Res 30:5382–5390
Leijonmarck M, Eriksson S, Liljas A (1980) Crystal structure of a
ribosomal component at 2.6 A resolution. Nature 286:824–826
Liao D, Dennis PP (1994) Molecular phylogenies based on ribosomal
protein L11, L1, L10, and L12 sequences. J Mol Evol 38:405–419
Maki Y, Hashimoto T, Zhou M, Naganuma T, Ohta J, Nomura T,
Robinson CV, Uchiumi T (2007) Three binding sites for stalk
protein dimers are generally present in ribosomes from archaeal
organism. J Biol Chem 282:32827–32833
Mears JA, Cannone JJ, Stagg SM, Gutell RR, Agrawal RK, Harvey
SC (2002) Modeling a minimal ribosome based on comparative
sequence analysis. J Mol Biol 321:215–234
Mohr D, Wintermeyer W, Rodnina MV (2002) GTPase activation of
elongation factors Tu and G on the ribosome. Biochemistry
41:12520–12528
Mulder FA, Bouakaz L, Lundell A, Venkataramana M, Liljas A,
Akke M, Sanyal S (2004) Conformation and dynamics of
ribosomal stalk protein L12 in solution and on the ribosome.
Biochemistry 43:5930–5936
Nissen P, Hansen J, Ban N, Moore PB, Steitz TA (2000) The
structural basis of ribosome activity in peptide bond synthesis.
Science 289:920–930
Noller HF (2004) The driving force for molecular evolution of
translation. RNA 10:1833–1837
Nomura T, Nakano K, Maki Y, Naganuma T, Nakashima T, Tanaka I,
Kimura M, Hachimori A, Uchiumi T (2006) In vitro reconstitution of the GTPase-associated centre of the archaebacterial
ribosome: the functional features observed in a hybrid form with
Escherichia coli 50S subunits. Biochem J 396:565–571
Olsen GJ, Woese CR (1997) Archaeal genomics: an overview. Cell
89:991–994
Page RD (1996) TreeView: an application to display phylogenetic
trees on personal computers. Comput Appl Biosci 12:357–358
123
J Mol Evol (2008) 67:154–167
Petoukhov MV, Svergun DI (2005) Global rigid body modeling of
macromolecular complexes against small-angle scattering data.
Biophys J 89:1237–1250
Petoukhov MV, Eady NA, Brown KA, Svergun DI (2002) Addition of
missing loops and domains to protein models by X-ray solution
scattering. Biophys J 83:3113–3125
Philippe H, Forterre P (1999) The rooting of the universal tree of life
is not reliable. J Mol Evol 49:509–523
Ramirez C, Shimmin LC, Newton CH, Matheson AT, Dennis PP
(1989) Structure and evolution of the L11, L1, L10, and L12
equivalent ribosomal proteins in eubacteria, archaebacteria, and
eucaryotes. Can J Microbiol 35:234–244
Remacha M, Jimenez-Diaz A, Bermejo B, Rodriguez-Gabriel MA,
Guarinos E, Ballesta JP (1995) Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the
pattern of protein expression in Saccharomyces cerevisiae. Mol
Cell Biol 15:4754–4762
Rodnina MV, Wintermeyer W (2001) Fidelity of aminoacyl-tRNA
selection on the ribosome: kinetic and structural mechanisms.
Annu Rev Biochem 70:415–435
Rodnina MV, Stark H, Savelsbergh A, Wieden HJ, Mohr D,
Matassova NB, Peske F, Daviter T, Gualerzi CO, Wintermeyer
W (2000) GTPases mechanisms and functions of translation
factors on the ribosome. Biol Chem 381:377–387
Rodriguez-Gabriel MA, Remacha M, Ballesta JP (2000) The RNA
interacting domain but not the protein interacting domain is
highly conserved in ribosomal protein P0. J Biol Chem
275:2130–2136
Santos C, Ballesta JP (1995) The highly conserved protein P0
carboxyl end is essential for ribosome activity only in the
absence of proteins P1 and P2. J Biol Chem 270:20608–20614
Shimizu T, Nakagaki M, Nishi Y, Kobayashi Y, Hachimori A, Uchiumi T
(2002) Interaction among silkworm ribosomal proteins P1, P2 and
P0 required for functional protein binding to the GTPase-associated
domain of 28S rRNA. Nucleic Acids Res 30:2620–2627
Shimmin LC, Ramirez C, Matheson AT, Dennis PP (1989) Sequence
alignment and evolutionary comparison of the L10 equivalent
and L12 equivalent ribosomal proteins from archaebacteria,
eubacteria, and eucaryotes. J Mol Evol 29:448–462
Soares MR, Bisch PM, Campos de Carvalho AC, Valente AP,
Almeida FC (2004) Correlation between conformation and
antibody binding: NMR structure of cross-reactive peptides from
T. cruzi, human and L. braziliensis. FEBS Lett 560:134–140
Sodeoka M, Larson CJ, Chen L, LeClaira KP, Verdine GL (1993) A
multifunctional plasmid for protein expression by ECPCR:
overproduction of the p50 subunit of NF-jB Bioorg. Med Chem
Lett 3:1089–1095
Svergun DI (1992) Determination of the regularization parameter in
indirect transform methods using perceptual criteria. J Appl
Crystallogr 25:495–503
Svergun DI (1993) A direct indirect method of small-angle scattering
data treatment. J Appl Crystallogr 26:258–267
Svergun DI, Petoukhov MV, Koch MH (2001) Determination of
domain structure of proteins from X-ray solution scattering.
Biophys J 80:2946–2953
Tchorzewski M (2002) The acidic ribosomal P proteins. Int J
Biochem Cell Biol 34:911–915
Tchorzewski M, Boguszewska A, Dukowski P, Grankowski N
(2000a) Oligomerization properties of the acidic ribosomal Pproteins from Saccharomyces cerevisiae: effect of P1A protein
phosphorylation on the formation of the P1A-P2B heterocomplex. Biochim Biophys Acta 1499:63–73
Tchorzewski M, Boldyreff B, Issinger O, Grankowski N (2000b)
Analysis of the protein-protein interactions between the human
J Mol Evol (2008) 67:154–167
acidic ribosomal P-proteins: evaluation by the two hybrid
system. Int J Biochem Cell Biol 32:737–746
Tchorzewski M, Krokowski D, Boguszewska A, Liljas A, Grankowski N (2003) Structural characterization of yeast acidic
ribosomal P proteins forming the P1A-P2B heterocomplex.
Biochemistry 42:3399–3408
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG
(1997) The CLUSTAL_X windows interface: flexible strategies
for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Res 25:4876–4882
Uchiumi T, Hori K, Nomura T, Hachimori A (1999) Replacement of
L7/L12.L10 protein complex in Escherichia coli ribosomes with
the eukaryotic counterpart changes the specificity of elongation
factor binding. J Biol Chem 274:27578–27582
Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape
determination in small angle scattering. J Appl Crystallogr
36:860–864
167
Wahl MC, Moller W (2002) Structure and function of the acidic
ribosomal stalk proteins. Curr Protein Pept Sci 3:93–106
Wahl MC, Bourenkov GP, Bartunik HD, Huber R (2000) Flexibility,
conformational diversity and two dimerization modes in complexes of ribosomal protein L12. EMBO J 19:174–186
Walsh DA, Doolittle WF (2005) The real ‘domains’ of life. Curr Biol
15:R237–R240
Woese C (1998) The universal ancestor. Proc Natl Acad Sci USA
95:6854–6859
Woese CR, Fox GE (1977) The concept of cellular evolution. J Mol
Evol 10:1–6
Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system
of organisms: proposal for the domains Archaea, Bacteria, and
Eucarya. Proc Natl Acad Sci USA 87:4576–4579
123