From the Department of Molecular Biology, University
of Aarhus, DK8000 Aarhus, Denmark and the
Department of
Chemistry and Biochemistry, Institute for Cellular and Molecular
Biology, University of Texas, Austin, Texas 78712
Received for publication, December 19, 2002, and in revised form, February 4, 2003
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ABSTRACT |
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The 18-kDa Domain I from the N-terminal region of
translation initiation factor IF2 from Escherichia
coli was expressed, purified, and structurally characterized
using multidimensional NMR methods. Residues 2-50 were found to form a
compact subdomain containing three short The initiation step of protein biosynthesis is
rate-limiting and hence an important point of regulation. In bacteria,
translation initiation is promoted by three protein factors:
IF1,1 IF2, and IF3. These
protein factors are essential in ultimately assembling the 30 S and 50 S subunits of the ribosome, the initiator fMet-tRNA The primary structure of initiation factor IF2 from different organisms
can be divided into distinct regions based on interspecies amino acid
sequence homology (5), as shown in Fig.
1. The C-terminal region of the protein
is highly conserved among species. This part has several functions
including a binding site for
fMet-tRNA-strands and three
-helices, folded to form a
motif with the three
helices packed on the same side of a small twisted
-sheet. The
hydrophobic amino acids in the core of the subdomain are conserved in a
wide range of species, indicating that a similarly structured motif is
present at the N terminus of IF2 in many of the bacteria. External to
the compact 50-amino acid subdomain, residues 51-97 are less conserved
and do not appear to form a regular structure, whereas residues 98-157
form a helix containing a repetitive sequence of mostly hydrophilic
amino acids. Nitrogen-15 relaxation rate measurements provide evidence
that the first 50 residues form a well ordered subdomain, whereas other regions of Domain I are significantly more mobile. The compact subdomain at the N terminus of IF2 shows structural homology to the
tRNA anticodon stem contact fold domains of the methionyl-tRNA and
glutaminyl-tRNA synthetases, and a similar fold is also found in the B5
domain of the phenylalanine-tRNA synthetase. The results of the
present work will provide guidance for the design of future experiments
directed toward understanding the functional roles of this widely
conserved structural domain within IF2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic diagram of E. coli
IF2. The protein encoded by the infB gene is
described with the positions of the alternative initiation sites
indicated. Full-length E. coli IF2 is referred to as IF2-1;
infB gene products that begin on the alternate initiation
sites are referred to as IF2-2 and IF2-3. Domains IV-VI are widely
conserved in all three phylogenetic kingdoms, whereas Domains I-III are
more variable in primary structure between species. A ribbon
diagram of the M. thermoautothrophicum aIF5B structure
derived from PDB entry 1G7R is shown (10); the structure of this
archaeal protein is homologous to Domains IV, V, and VI of the E. coli IF2. The present study focuses on the region of
E. coli IF2-1 that precedes the first alternate initiation
site, consisting of the residues up to 157.
Bacterial IF2 is encoded by the infB gene, which in Escherichia coli encodes three forms of IF2: IF2-1, -2, and -3, of molecular masses 97.3, 79.9, and 78.8 kDa, respectively (12). The expression of IF2-2 and IF2-3 in E. coli is by tandem translation of the intact infB mRNA, and not by translation of post-transcriptionally truncated mRNA. Hence, the three different forms of IF2 have identical C termini (13). The presence of both the large and smaller forms is required for optimal growth of E. coli. The cellular content of IF2-2 and -3 is close to the level of IF2-1 (14, 15). The presence of more than one isoform of IF2 is not a phenomenon peculiar to E. coli, but has been found in several other enterobacteria (16).
The N-terminal region of IF2 differs from the C-terminal region in that there is significantly more variability between species in primary structure as well as length. We have previously used sequence data and biochemical experiments to divide the N-terminal region of E. coli IF2 into three separate domains designated Domain I, II, and III (17), as illustrated in Fig. 1. A function for the domains in the N-terminal region has been demonstrated in E. coli, where a fragment of IF2 consisting of Domains I and II, but not a fragment consisting of Domain I alone, binds to the 30 S ribosomal subunit (18, 19). Furthermore, we have recently used a primer extension inhibition assay to identify Domains I-II of E. coli IF2 as an interaction partner for the infB mRNA (16).
The present work describes the results of nuclear magnetic resonance
(NMR) and circular dichroism (CD) experiments used to characterize the
18-kDa Domain I of E. coli IF2. A search among structures in
the Protein Data Bank revealed that this domain has no significant
primary sequence homology to any protein of known structure, and a
BLAST search (20) of the non-redundant protein sequence data available
at the National Center for Biotechnology Information (NCBI) Web site
showed that the domain has no significant sequence homologue other than
the same domain within IF2 of different species.
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EXPERIMENTAL PROCEDURES |
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Protein Cloning, Expression, and Purification--
The fragment
of the infB gene encoding the first domain of IF2-1 was
amplified by PCR using E. coli K12 as template and primers that included unique restriction sites for XbaI and
NdeI for insertion into the pET-15b expression vector
(Novagen). DNA sequencing confirmed the insertion of infB
into the vector. The protein was expressed in BL21(DE3) cells
(Novagen). Cells were grown in M9 minimal medium supplemented with 100 mg/liter ampicillin. Protein expression was induced with 0.1 mM isopropyl-1-thio--D-galactopyranoside when the cells reached an OD550 of 0.6. Cells from a
1-liter culture were harvested by centrifugation and dissolved in 20 ml
of buffer A (50 mM Hepes, pH 7.6, 10 mM
MgCl2, 1 mM dithiothreitol, 0.1 mM
phenylmethylsulfonyl fluoride, 15 mM NaN3). The
solution was passed once through a French pressure cell at 1500 PSI and
centrifuged at 30,000 × g for 1 h. The
supernatant was loaded on a 40-ml SP Sepharose FF column (Amersham
Biosciences), and bound protein was eluted with a 0-200 mM
NaCl step gradient. The buffer was changed to buffer A using a Sephadex
G25 column (Amersham Biosciences). The pooled fraction was passed
through a Source 30Q column, and the unbound protein was loaded on a
20-ml SP Sepharose HP column (Amersham Biosciences). The IF2 Domain I
was eluted with a gradient from 0 to 200 mM NaCl over 8 column volumes, yielding 40 mg of pure protein per 1 liter of culture
medium. The purified protein was subjected to N-terminal sequencing by
Edman degradation, and the protein mass was determined by MALDI-TOF
analysis. Samples of protein enriched in 15N or
15N and 13C simultaneously were prepared as
described above, but cells were grown in M9 minimal medium containing
1.5 g/liter [13C] glucose and/or 0.6 g/liter
[15N] ammonium chloride (Cambridge Isotope Laboratories)
as sources of carbon and nitrogen, respectively.
Circular Dichroism Spectroscopy-- The circular dichroism spectra were recorded on the UV1 photobiology synchrotron beamline at the Institute for Storage Ring Facilities at Aarhus University, Denmark, using synchroton radiation provided by the ASTRID storage ring. Spectra were recorded in 10 mM phosphate buffer, pH 6.0 using an open 0.01-mm Hellwa suprisil quartz cell. The data were acquired using 5 consecutive scans with 1-nm intervals in the range 180-250 nm. Spectra of each sample were recorded from 5 to 70 °C in 5 °C steps. The sample was allowed to equilibrate at each temperature for 20 min before acquiring the spectra. The data were normalized to a 1 mg/ml concentration in a 1-mm path length cell.
NMR Spectroscopy-- NMR spectra were recorded at 20, 30, and 40 °C using a 500 MHz Varian Inova spectrometer equipped with a triple-resonance probe and z-axis pulsed-field gradient. NMR samples typically contained 2-3 mM of the protein and 10 mM sodium phosphate in 90% H2O/10% D2O or 100% D2O solvent at pH 6.0. Backbone resonance assignments were obtained by analyzing HNCA, HNCO, HNCACB, HN(CO)CACB, and HACACBCO spectra, which correlate the backbone protons to the N, Ca, Cb, and CO signals of the same and adjacent amino acid residues. 15N-edited HSQC-TOCSY, 13C-edited HCCH-TOCSY, and two-dimensional 2QF-COSY and TOCSY spectra were used for side-chain resonance assignments. NOE cross-peaks were detected using two-dimensional 1H-1H NOESY, three-dimensional 15N-resolved 1H-1H HSQC-NOE, and three-dimensional 13C-edited 1H-1H HSQC-NOESY spectra. The 13C-edited 1H-1H NOE spectrum was collected in 90% H2O/10%D2O solvent, so that NOE peaks between amide and side-chain protons could be resolved by the chemical shift of a side-chain 13C nucleus. Rapidly exchanging amide protons were identified by comparing 15N-1H correlated spectra obtained with selective excitation versus presaturation for solvent suppression. Data were processed using either the program NMR-Pipe (21) or Felix 1.0 (Hare Research). 1H, 15N, and 13C chemical shifts are referenced as recommended by Ref. 22, with proton chemical shifts referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 0 ppm. The 0 ppm 13C and 15N reference frequencies were determined by multiplying the 0 ppm 1H reference by 0.251 449 530 and 0.101 329 118, respectively.
Structure Determination--
Structure calculations for IF2
Domain I were performed using the hybrid distance geometry-simulated
annealing and energy minimization protocols within the CNS version 1.1 program suite (23). Distance restraints were derived from
multidimensional NOE spectra. In order to minimize the effects of spin
diffusion, as many of the NOE cross-peaks as possible were identified
in homonuclear two-dimensional NOE spectra acquired with relatively
short mixing times (60 ms); these spectra also offered the best digital
resolution. Peaks from these short mixing time spectra were placed into
four categories: strong (<3.2 Å), medium (<3.6 Å), weak(< 4.2 Å),
and very weak (<4.6 Å). Additional NOE cross-peaks were identified in
the three-dimensional 15N- and 13C-edited NOE
spectra (60-ms mixing time) and assigned to distance restraints as
strong (<5.0 Å), medium (<5.5 Å), weak (<6.3 Å), and very weak
(<6.9 Å). A very conservative distance restraint of <7.9 Å was used
for NOE cross-peaks identified in spectra obtained with a relatively
long mixing time (160 ms), where the effects of spin diffusion are most
likely to be present. Pseudoatom corrections were included for NOEs
including stereospecifically unassigned methyl protons of Val or Leu,
where distances were measured from the center of the two methyl groups,
and 2.5 Å was added to the interproton distance. For NOEs involving
other methyl groups distances were measured from the center of the
methyl group, and 1.0 Å was added to the interproton distance.
Stereospecifically unassigned methylene groups were treated the same
way, and 0.7 Å was added to the interproton distance. For NOEs
involving and
protons of Phe, distances were measured from the
central point between the two atoms, and 2.4 Å was added to the
interproton distance. For regions of regular
-helix or
-strand
structure identified by characteristic NOE patterns and chemical shift
indices (CSI) (24), backbone torsion angle restraints were included for
the
and
angles. For
-strands,
and
angles were
restricted to
120o ± 25o and
150o ± 25o, respectively, and for
-helices
both
and
angles were restricted to
60o ± 25o. Hydrogen bond restraints were only included for amide
protons with relatively slow solvent exchange rates that are also
located in regions of regular
-helix or
-sheet structure. Twenty
diverse starting structures were generated by subjecting a random coil model to the CNS simulated annealing protocol using only the dihedral angle and hydrogen bond constraints. These structures were then used as
starting models for 200 runs of the simulated annealing protocol. Most
of the simulated annealing runs resulted in similar structures with
similar energies. From this final set of refined models, a set of 20 structures were selected that satisfy the following criteria: 1) their
CNS energy term is at or very near the minimum value obtained, 2) there
are no interproton distance constraint violations of greater than 0.5 Å, 3) the set of models are a fair representation of the full range of
structures that satisfy the NMR-derived restraints while having
reasonable molecular geometry, as defined by the CNS energy function.
Structural statistics (Table I) were calculated with the assistance of
the program PROCHECK-NMR (25).
15N NMR Relaxation Rates--
The 15N
T1 and T2 relaxation
times and the 15N-1H NOE were measured using
pulse sequences (26) that feature gradient selection and sensitivity
enhancement, and pulses for minimizing saturation of the solvent water.
Six two-dimensional spectra with relaxation delays of 10, 260, 510, 760, 1010, and 1260 ms were acquired for the T1
relaxation measurements, and six two-dimensional spectra with
relaxation delays of 29, 58, 87, 116, 145, and 174 ms were acquired for
the T2 relaxation measurements; in each case the relaxation delay between the acquisition of each free induction decay
was 3 s. The spectra for measuring the
15N-1H NOE were acquired with either a 5-s
delay between each free induction decay or a 1-s delay followed by a
4-s long series of 120o nonselective 1H pulses.
The T1 and T2 data were
fitted to a single exponential decay function of the form I = I0et/Td, in which I is the
intensity of the signal at time t, I0 is the intensity at time t = 0 and Td
is the decay constant T1 or
T2, respectively. Rotational correlation times
and order parameters were calculated using Modelfree 4.0 (27) as
previously described (28).
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RESULTS |
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Domain I of E. coli translation initiation factor IF2
was recombinantly expressed, and the purified domain found to be
soluble, stable, and well suited for study using biophysical methods.
MALDI-TOF mass spectrometry revealed that the N-terminal methionine
residue was post-translationally removed from the protein. CD spectra contain features typical of a protein with substantial -helical content (Fig. 2A), with
characteristic minima at 207 and 222 nm. CD spectra recorded at
30 °C or below look essentially the same, whereas spectra recorded
at higher temperatures differ significantly, presumably due to
unfolding of the protein (Fig. 2B). The circular dichroism
at 207 nm increases with increasing temperature, consistent with a
decrease in helical content. The presence of an isodichroic point in
the CD spectra indicates that the unfolding is a two-state process. The
protein can be reversibly denatured by heating; a sample heated to
70 °C and then cooled to 20 °C has a CD spectrum that is the same
as that recorded before heating of the sample. Two-dimensional NMR
spectra acquired at 20 and 30 °C are essentially identical and of
excellent quality, with the majority of the resonances being well
dispersed, as is typical for a folded protein (Fig. 3). However, most NOE cross-peaks were
absent in spectra acquired at 40 °C. The NMR and CD data are
therefore consistent in indicating that the domain starts to lose
structure between 30 and 40 °C.
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An abundance of inter-residue NOE cross-peaks were observed for
residues 2-50 of Domain I, consistent with these residues forming a
compact globular subdomain. In contrast, residues 51-157 only exhibit
NOE cross-peaks between pairs of protons that are relatively close
together in the primary sequence; these residues are therefore likely
to form a linker region connecting the compact folded structure formed
by amino acids 2-50 with the other domains of IF2. The structural
details of each of these subdomains within Domain I of IF2 will be
discussed in turn. A summary of local NOE patterns and CSI, which
define the secondary structure of the protein are given in Fig.
4.
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Structure of a Conserved Subdomain (Residues 2-50)--
The
structure of residues 2-50 at the N terminus of IF2 was determined
from distance constraints derived from observed NOE intensities, and
torsion angle and hydrogen bond constraints derived for the regions
identified as having regular -sheet or helical structure. Complete
backbone and nearly complete side chain chemical shift assignments were
obtained for the 1H, 13C, and 15N
nuclei in the subdomain. Of particular significance, complete 1H resonance assignments were obtained for all of the
leucine, isoleucine, valine, phenylalanine, and alanine side chains
(among others) in the subdomain; assignment of NOE cross-peaks derived from these side chains were critical in defining the hydrophobic core.
A superposition of a set of structures that are equally consistent with
more than 900 NMR-derived constraints is shown in Fig.
5. These structures are a fair
representation of the full range of structures that are consistent with
the NMR data. Structural statistics for residues 2-50 are summarized
in Table I. Coordinates for the subdomain
have been deposited in the Protein Data Bank (PDB), where it has been
assigned accession number 1ND9.
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The NMR results show that the first 50 amino acids of IF2 form a
compact structure consisting of three -strands and three short
-helices. The three helices are nearly orthogonal, and are located
on the same side of an antiparallel twisted sheet formed by three
-strands. Strands
1 (residues 3-6) and
2 (residues 29-32)
are linked by helices
1 (residues 8-12) and
2 (residues 16-26),
strand
3 (residues 35-39) is connected to
2 by a short loop, and
the compact subdomain terminates with helix
3 (residues 42-50).
Alignments of IF2 sequences from different species show that
hydrophobic residues Ile-6, Leu-9, Val-17,
Leu-20, Val-21, Phe-24, Ala-27, Ile-29,
Val-37, Leu-45, Ile-46, and Leu-49 are conserved in a wide range of
species (Figs. 6 and 7); these residues are all buried and form the core of the subdomain structure. A ribbon diagram depicting the fold of the subdomain is shown
in Fig. 5.
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In previous work, monoclonal antibodies were generated against IF2 from
E. coli (17). One of these antibodies was epitope mapped on
native full-length IF2 to the region of 2-
3 and the loop
connecting these strands, suggesting that this region may be
solvent-exposed in the full-length IF2; however, we note that epitope
mapping is not an unambiguous indicator of solvent-exposed residues.
The coordinates for the structure of the IF2 N-terminal subdomain were
compared against a data base of known structures using the Vector
Alignment Search Tool (VAST), located at the NCBI Web page, and the
DALI search tools (29). The subdomain was found to be structurally
similar to the B5 motif within Phe-tRNA synthetase (Fig.
8); the structures share the same
topology, and the rmsd of the backbone atoms is 3.3 Å. The
1-strand and the three helices (
1,
2, and
3) in the
domain of IF2 also superimpose well with a domain of methionyl-tRNA
synthetase and glutaminyl-tRNA synthetase known as the stem contact
(SC) fold (30). The rmsd of the backbone between the SC fold domains in
the E. coli aminoacyl tRNA synthetases and the secondary
structure elements
1,
1,
2, and
3 of the subdomain of IF2
are 2.4 and 2.6 Å, respectively (Fig. 8).
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Structure of the Less Conserved Region (Residues
51-97)--
Residues 51-97 are not as widely conserved as residues
2-50, as indicated in Fig. 6. Although chemical shifts were assigned for most nuclei (all those except residues 63-67, 88, and 97), the
large majority of chemical shifts of the backbone as well as side-chain
atoms appear at or very near to the random coil values, even for the
very hydrophobic side chains. The only identified NOEs were
intraresidue, sequential, and in a few cases medium range (Fig. 4).
Without any observed long range NOEs, the three-dimensional structure
of this region of the protein cannot be determined from the NMR data.
Local NOE patterns, as well as C, CO, and
H
chemical shifts indicate that residues Val-83-Val-85
are likely to be in an extended (
-strand) conformation, however no
long range NOEs to connect this strand to any region of the N-terminal subdomain were found. We conclude that residues 51-97 do not have a
well defined structure under the conditions used for the NMR experiments. However, it is possible that these residues are structured in the context of the full-length IF2.
Structure of the C-terminal Region of Domain I (Residues
98-157)--
The 60-residue C-terminal region of IF2 Domain I
contains a repetitive amino acid sequence where every fourth residue is
an alanine, and the alanines are separated by sequences rich in
glutamate, glutamine, lysine, and arginine. The pattern Arg-Glu-Ala is
repeated six times. The repetitive sequence made chemical shift
assignments particularly challenging for this region of the molecule.
Unambiguous sequence specific assignments were obtained for residues
98-104, 120-126, 149-157; these residues exhibit the sequential and
medium range NOE patterns indicative of -helical conformation, as
well as C
, CO, and H
chemical
shifts typical of helical structure (Fig. 4). Another helical
Ala-Ala-Glu unit was resolved but could not be placed unambiguously in
the sequence. Unresolved, overlapping peaks observed in the
triple-resonance spectra at the chemical shifts expected for arginine,
glutamate, and glutamine in
-helical conformation probably account
for the resonances of the remaining residues in the C-terminal region
of Domain I.
The 60-residue helix at the C terminus of Domain I has the potential to form a coiled-coil structure, resulting in dimerization of the protein. Analytical ultracentrifugation was therefore used to test for the presence of a homodimeric structure. The sedimentation coefficients of IF2 Domain I (residues 2-157), hen egg white lysozyme (129 residues), and bovine carbonic anhydrase (259 residues) were determined after being dialyzed against identical solutions (1 mM phosphate buffer, pH 6, 20 °C), the sedimentation coefficients of the three proteins were found to be 1.46 S, 1.85 S, and 2.85 S, respectively. The observation that Domain I has a sedimentation coefficient that is low for its molecular weight (and less than that of lysozyme) can be best explained by the domain being a monomer with a significantly non-spherical shape. Consistent with the ultracentrifugation results, a half-filter nuclear Overhauser effect experiment designed to detect inter-subunit NOEs in a mixture of unlabeled and 13C/15N-labeled protein (31) provided no evidence for dimerization.
The mostly hydrophilic nature of residues 98-157 suggests that this
helical structure is solvent exposed, perhaps forming a linker
connecting Domain I with the other domains of IF2 similar to the
helical linker that connects Domain VI-1 with VI-2 in IF2 (Fig. 1).
Earlier epitope mapping studies of monoclonal antibodies on native IF2
from E. coli identified two non-overlapping epitopes in the
region of residues 108-137 (17); this provides some independent evidence supporting the hypothesis that these residues are
solvent-exposed in the full-length IF2 (however, it is again noted that
epitope mapping is not an unambiguous indicator of solvent exposure). Although the precise amino acid sequence of residues 98-157 is not
conserved, many of the bacteria contain similar repetitive sequences of
mostly hydrophilic amino acids with a high propensity for forming an
-helix (Fig. 6), suggesting that the helical linker may be a feature
that is present in Domain I of IF2 of many of the bacteria.
15N Relaxation Rates and Internal Motions within Domain
I of IF2--
15N relaxation rate data
(T1, T2, and
15N-1H NOE) were obtained for 48 backbone amide
nitrogens of residues in the range 6-157 that have resonances that are
well resolved in two-dimensional 15N-1H
correlated spectra. In terms of 15N-1H
relaxation rates, the domain can be divided into three regions. For
residues 6-50, the observed values for T1,
T2 and the 15N-1H NOE
are strikingly uniform, averaging 0.54 s, 0.14 s, and 0.62, respectively. These values are consistent with a well ordered structure
with a rotational correlation time of 6.7 ns. This rotational correlation time is typical of protein with a molecular weight significantly less than that of the full 156 residue Domain I, and
therefore suggests that the first 50 residues form a relatively rigid
subdomain that moves independently of the other regions of the protein.
Residues 55-95 exhibit negative values for the 15N-1H NOE and relatively long
T2 relaxation times, indicating a flexible and
disordered structure. For the long helix at the C terminus of Domain I,
only four residues (123, 149, 152, and 157) have amide resonances that
are well enough resolved for relaxation rate data to be obtained. These
residues differed widely in their relaxation rates, suggesting that the
motions of the long helix cannot be described using a simple model.
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DISCUSSION |
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How well conserved is the compact /
subdomain that we have
identified at the N terminus of IF2? To systematically address the
issue of structural conservation, the amino acid sequences at the N
termini of IF2 were examined for a set of 68 diverse bacteria whose
genomes have been sequenced. IF2 in 49 of 68 bacteria contains an
N-terminal sequence that is clearly homologous to the
/
motif at
the N terminus of E. coli IF2, as indicated by the strong
conservation of the residues that form the hydrophobic core of the
subdomain (Fig. 7). IF2 in 13 of the 68 organisms may contain a
homologous N-terminal structure; in these cases it was difficult to be
certain, since aligning the sequences required the insertion of one or
more gaps. Only 6 of 68 species clearly do not appear to contain a
sequence homologous to the 50-amino acid N-terminal motif. An example
of these organisms is Mycoplasma genitalium, the bacterium
with the smallest known genome; in this species IF2 is unusually small,
containing only 620 amino acids rather than the ~900 amino acids
found in IF2 of most bacteria. In summary, our investigation of the
amino acid sequences of IF2 in various species indicates that the large
majority of the bacteria contain a 50 amino acid
/
motif at the N terminus of their IF2 that is structurally homologous to
that found in E. coli.
Although the conserved hydrophobic residues indicate that the overall
shape of the 50-amino acid subdomain is well conserved across a wide
range of bacteria, it is interesting that there are no surface residues
that are as well conserved as the hydrophobic core. For example, the
surface residues at positions 18, 41, and 42 in the and
proteobacteria are conserved in terms of their charge (indicated by
black arrows in Fig. 6), however, residues at these same
positions are not conserved in a broader range of bacterial species
(Fig. 7). Conversely, there are other surface residues that are
conserved in a wide range of bacteria (such as lysine 3 in
Bacillus subtilis, Fig. 7) that differ in the
and
proteobacteria (Fig. 6). The strong conservation of the subdomain
structure suggests its general importance, while the variability of its
surface residues could be explained by its precise function being more
species-specific.
The present data indicate a structural relationship between the
N-terminal subdomain and the SC fold domains in the class Ia aminoacyl
tRNA synthetases. The SC fold domain docks to the inner side of the
L-shaped tRNA, thereby positioning the anticodon stem of the tRNA (30).
The domain connects the acceptor and anticodon binding domains of the
tRNA synthetases and may provide a functional communication of
anticodon recognition between the anticodon binding domain to the
acceptor region binding domain and active site of the synthetases (32).
Interestingly, even though a high degree of structural homology is
found for the fold in the Gln- and Met-tRNA synthetases, no significant
sequence similarity exists, although generally the C-terminal helix in the motif has a net negative charge despite its location adjacent to
the highly negatively charged phosphate backbone of the tRNA (33). A
similar pattern is found in the case of IF2, where there is no obvious
sequence homology for the surface residues of the subdomain, but a net
negative charge is found for the region corresponding to helix 3 of
the subdomain in the great majority of bacterial sequences investigated.
IF2 interacts with the initiatior
fMet-tRNA
Further studies are clearly required to fully characterize the
structural and functional properties of the N terminus of IF2, and to
address the puzzling question of why more than one isoform of IF2,
differing only in the presence of Domain I, exists in the
enterobacteria. The structural view of Domain I provided by the present
work will aid in the design of experiments directed toward determining
more specifically the details of the interactions between Domains I-II
of IF2, the 30 S ribosomal subunit, and the infB mRNA.
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ACKNOWLEDGEMENTS |
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We thank John Kenney and Cedric Dicko at the Institute for Storage Ring Facilities, Aarhus University, Denmark for help with Circular Dichroism.
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FOOTNOTES |
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* This work was funded by Grants 9901722 and 51-00-0263 from the Familien Hede Nielsens Fund and the Danish Natural Science Research Council (to H. U. S.-P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Chemical shift assignments for the domain of IF2 have been submitted to the BioMagResBank and assigned the accession number BMRB-5624.
The atomic coordinates and the structure factors (code 1ND9) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Supported by a personal grant from Knud Højgaards Fund, Denmark.
¶ To whom correspondence may be addressed: Dept. of Molecular Biology, University of Aarhus, Denmark. Tel.: 45-89425050; Fax: 45-86182812; E-mail: husp@biobase.dk.
** Supported by Grant F-1353 from the Welch Foundation. To whom correspondence may be addressed: Dept. of Chemistry and Biochemistry, Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712. Tel.: 512-471-7859; Fax: 512-471-8696; E-mail: dhoffman@mail.utexas.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M212960200
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ABBREVIATIONS |
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The abbreviations used are: IF, initiation factor; rmsd, root mean square deviation; SC, stem contact; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; CSI, chemical shift index; PDB, protein data bank.
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