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INTRODUCTION |
Guanine nucleotide-binding proteins play crucial roles in numerous
cellular processes (1). These factors cycle through GDP- and GTP-bound
states that reflect their active and inactive forms. Many of these
proteins require a guanine nucleotide exchange factor to promote the
conversion of the GDP-bound form to the GTP-bound form. One of the best
studied of the guanine nucleotide-binding proteins is elongation factor
Tu (EF-Tu).1 During the
process of polypeptide chain elongation in protein biosynthesis, EF-Tu
promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site
(A-site) of the ribosome (2). This binding reaction requires the
formation of a ternary complex (EF-Tu·GTP·aa-tRNA). The correct
ternary complex is selected through codon-anticodon interactions at the
A-site. EF-Tu then hydrolyzes the bound GTP and is released from the
ribosome as an EF-Tu·GDP complex (3). A second elongation factor
(EF-Ts) promotes the release of GDP forming an intermediate EF-Tu·Ts
complex (4). In Escherichia coli, GTP binds to the
EF-Tu·Ts complex promoting the release of EF-Ts and the formation of
an EF-Tu·GTP complex. This complex binds another aa-tRNA forming the
ternary complex again and the cycle repeats.
The three-dimensional structures of EF-Tu complexed to its ligands have
been reported (5-9). Analysis of these structures indicates that EF-Tu
folds into three domains. Domain I encompasses the first 200 residues
and includes the guanine nucleotide-binding site. Domains II and III
are each about 100 residues in length. All three domains are required
for binding the aa-tRNA (8). EF-Ts can be divided into four structural
units as follows: the N-terminal domain (residues 1-54), the core
domain (residues 55-179 and 229-263), the dimerization domain
(residues 180-228), and the C-terminal module (residues 264-282). The
core domain is divided into subdomain N (residues 55-140) and
subdomain C (residues 141-179 and 229-263). The crystal structure
indicates that there are extensive regions of contact between E. coli EF-Tu and EF-Ts (9). The N-terminal domain, subdomain N, and
the C-terminal module all interact with domain I of EF-Tu, whereas
subdomain C interacts with domain III.
Examination of the three-dimensional structure of the EF-Tu·Ts
complex originally suggested that nucleotide exchange arises in part
because the side chains of Asp-80 and Phe-81 of EF-Ts intrude into a
site on EF-Tu near where the Mg2+ ion interacting with GDP
is normally located (9). The resulting disruption of the
Mg2+ ion binding site was postulated to reduce the affinity
of EF-Tu for GDP. However, mutation of Asp-80 or Phe-81 to Ala only
results in a 2-3-fold reduction in the ability of EF-Ts to catalyze
guanine nucleotide exchange with EF-Tu·GDP (10). The mutation of both Asp-80 and Phe-81 has a more deleterious effect (about 10-fold) on the
activity of E. coli EF-Ts. These observations suggest that other regions of EF-Ts must be playing a role in promoting GDP exchange
and/or in the binding of EF-Ts to EF-Tu. In the present work, we have
prepared a number of mutations that alter resides at the interface
between EF-Tu and EF-Ts. The effects of these mutations on the affinity
of EF-Ts for EF-Tu and on its ability to catalyze guanine nucleotide
exchange have been analyzed.
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MATERIALS AND METHODS |
Construction of E. coli EF-Tu and EF-Ts Mutants--
E.
coli EF-Tu and EF-Ts genes were cloned into pET24c(+) (10), and
site-directed mutagenesis of individual residues was performed using a
polymerase chain reaction-based "linker scanning" method (11).
Polymerase chain reaction was also used to construct a derivative of
EF-Ts carrying a deletion in helix h13 (residues 271-282).
Expression and Purification of EF-Tu and EF-Ts--
A His-tagged
form of E. coli EF-Tu was expressed and purified as
described previously (10). A His-tagged form of E. coli EF-Ts was purified under two different conditions basically as described previously (10). In the first set of conditions, no GDP was
present in the extraction buffers. This procedure was used to analyze
the amount of EF-Tu co-purifying with EF-Ts or it mutated derivatives.
In the second set of conditions, 10 µM GDP was included
in the buffers as described previously (10). This procedure was used to
prepare EF-Ts or its variants free of EF-Tu.
Measurements of Binding Constants and Assays of
Activities--
The equilibrium association constants for the binding
of GDP to mutated forms of EF-Tu were determined as described
previously (12). The binding constants governing the interaction of
various mutated forms of E. coli EF-Ts to EF-Tu were
determined basically as described previously (13). The binding
constants governing the interaction of various mutated forms of
E. coli EF-Tu to wild-type EF-Ts were determined in a
similar manner (13). For these experiments, EF-Tu·GDP (0.5 µM, 50 pmol), [3H]GDP (500 cpm/pmol) (5 or
10 µM), and EF-Ts or its variants (1-3 µM,
100-300 pmol) were incubated in a reaction mixture (100 µL) at
20 °C for 30 min as described (13). The amount of EF-Tu·GDP at
equilibrium was measured by the nitrocellulose filter binding assay
(14). Kobs for Reaction 1 is shown below in
Equation 1.
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(Reaction 1)
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(Eq. 1)
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Kobs was calculated in each case and used
to determine the binding constant (KTs) for
Reaction 2
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(Reaction 2)
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using the relationship shown in Equation 2.
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(Eq. 2)
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Where KTu·GDP is the binding constant
of EF-Tu to GDP (3.3 × 108
M
1) (12). This value for
KTu·GDP was used for experiments using
wild-type EF-Tu and for the H19A and E348A derivatives. KTu·GDP was determined to be 7.9 ± 2.1 × 107 for the Q114A variant of EF-Tu. The
wild-type EF-Tu and the E348A derivative used for these experiments
were about 50% active based on their abilities to bind GDP. The Q114A
and the H19A variants of EF-Tu were estimated to be 20% active as
estimated by their ability to bind GDP at saturating concentrations of
the nucleotide. EF-Ts was estimated to be fully active based on the
percentage that could bind to EF-Tu. The activities of EF-Ts and its
variants in promoting GDP exchange with EF-Tu·GDP and in stimulating
the activity of this factor in poly(U)-directed polymerization of phenylalanine were determined as described (14, 15).
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RESULTS |
Mutational Analysis of EF-Ts Residues Involved in the Interactions
between the N-terminal Domain of EF-Ts and Domain 1 of EF-Tu--
The
N-terminal domain of EF-Ts makes a number of contacts with domain I of
EF-Tu (Fig. 1A). These
interactions include Ala-5, Lys-9, Arg-12, Met-19, Met-20, and Arg-23
of EF-Ts. This area of the interface is bound on the edges by
electrostatic and H bond contacts, whereas the interior of the surface
is predominantly hydrophobic. This type of distribution of hydrophilic
and hydrophobic contacts on the surface of a protein-protein
interaction site is quite common (16, 17).

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Fig. 1.
Residues of EF-Tu and EF-Ts in contact.
A, residues in the N-terminal domain of EF-Ts contacting
residues in domain I of EF-Tu. Arg-12 contacts both Glu-152 and the
backbone carbonyl of Asp-109. Only the former interaction is shown for
the sake of simplicity. B, contacts between residues in
subdomain N of the core of EF-Ts with domain I of EF-Tu. C,
residues in subdomain C of the core of EF-Ts in contact with domain III
of EF-Tu. Displayed using RASMOL (21). Oxygens are shown in
red; nitrogens are shown in purple, and sulfur
atoms are shown in yellow.
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To assess the relative importance of the interactions of these residues
in EF-Ts with domain I of EF-Tu, four mutants of E. coli
EF-Ts (K9AR12A, M19AM20A, M19EM20E, and K23A) were constructed and
purified from E. coli as His-tagged proteins. When cell
extracts are prepared in buffers containing Mg2+ but
lacking GDP, small amounts of EF-Tu are present when wild-type EF-Ts is
expressed (data not shown). This level of EF-Tu (about 1 mol of EF-Tu
per 10-20 mol of EF-Ts) reflects the relative ability of EF-Ts to
compete for binding to EF-Tu with the guanine nucleotides and aa-tRNA
present in the cell extract. When cell extracts are prepared in buffers
containing GDP, no EF-Tu is present in the preparations of the
wild-type factor or of any of the mutated forms described below ((10)
and data not shown).
The first mutated form of EF-Ts to be tested was K9AR12A. Surprisingly,
Lys-9 interacts with EF-Tu primarily through a hydrophobic contact
between a side chain ---CH2--- and Leu-148 in domain I of EF-Tu. Arg-12 makes an electrostatic interaction with Glu-152 and a
hydrogen bond with the backbone of Asp-109 in EF-Tu (Fig. 1A). No EF-Tu co-purifies with the K9AR12A derivative of
EF-Ts (data not shown). This observation suggests that one or both of these residues is important for the interaction of EF-Ts with EF-Tu. To
assess the role of these two residues more fully, an attempt was made
to measure the binding constant between EF-Tu and the K9AR12A
derivative of EF-Ts. These measurements are made by determining the
ability of EF-Ts to compete with GDP for binding to EF-Tu (12, 13). The
interaction of wild-type EF-Ts with EF-Tu is characterized by a binding
constant of about 9 × 108
M
1 (Table I).
The binding constant for the K9AR12A derivative is too weak to be
measured in the competition assay used suggesting that there is at
least a 100-fold reduction in this value. This mutated form of EF-Ts
has no detectable activity in promoting guanine nucleotide exchange
with EF-Tu (Fig. 2A) or in
stimulating the activity of this factor in poly(U)-directed
polymerization of phenylalanine (Fig. 2B).

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Fig. 2.
A, activities of derivatives of EF-Ts
carrying mutations in the N-terminal portion of the factor. The
stimulation of GDP exchange with EF-Tu was examined as described under
"Materials and Methods." Reaction mixtures contained 7.3 µg of
expressed EF-Tu (about 80 pmol of active factor) and the indicated
amounts of EF-Ts or its mutated derivatives. Blanks representing the
amount of GDP exchange carried out by EF-Tu alone during this
incubation period (about 7 pmol) have been subtracted from each value.
No GDP binding could be detected in the absence of EF-Tu indicating
that the preparations of EF-Ts used here were free of EF-Tu.
B, stimulation of the activity of E. coli EF-Tu
in poly(U)-directed polymerization by EF-Ts and its mutants. Reaction
mixtures contained 1 pmol of expressed EF-Tu and the indicated amount
of EF-Ts. Blanks representing the amount of polymerization catalyzed by
EF-Tu alone (about 6 pmol) have been subtracted from each value.
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It is always possible that a mutation resulting in an inactive protein
exerts its effect by preventing the normal folding of the polypeptide
chain. To assess whether the lack of activity observed with the K9AR12A
derivative is the result of the failure of the protein to fold, CD
spectroscopy was carried out on wild-type EF-Ts and on the K9AR12A
derivative. Spectra were collected from 178 to 260 nm allowing an
assessment of the ability of the mutated protein to fold into the
correct secondary structure. The CD spectra of this mutated protein is
identical to that of the wild-type protein indicating that it has
folded correctly (data not shown).
The observations above indicate that Lys-9, Arg-12, or both of these
residues make an essential contact with EF-Tu that is important for the
strength of the interaction between these two factors and for the
biological activity of EF-Ts. In assessing the relative importance of
Lys-9 and Arg-12 in EF-Ts, it should be noted that the major contact
between Lys-9 and domain I of EF-Tu is through the
-methylene group
that is about 3.8 Å from the side chain of Leu-148. This contact is
still available when Lys-9 is mutated to Ala. An examination of the
conservation of this residue in EF-Ts from various organisms indicates
that Lys-9 is generally a Lys residue in prokaryotes but is Met in
mitochondrial EF-Ts. In contrast, Arg-12 is 100% conserved in the
EF-Ts' that have been sequenced to date. All of these observations
indicate that the lack of activity of the K9AR12A derivative is
probably due to the mutation of Arg-12 suggesting that this residue
plays a critical role in EF-Ts.
The central core of the interactions between the N-terminal domain of
EF-Ts and domain I of EF-Tu is formed by Met-19 and Met-20 which make
hydrophobic contacts with Val-140, Leu-145, Leu-148, and Val-149 of
EF-Tu (Fig. 1A). They also make backbone hydrogen bonds with
residues in domain I of EF-Tu. Although methionine is not universally
present at positions 19 and 20 of EF-Ts, these residues are generally
hydrophobic as would be expected. When a mutated derivative of these
residues (M19AM20A) is prepared and purified from E. coli, a
small amount of EF-Tu can be seen co-purifying with this protein. In
contrast, no EF-Tu is observed in preparations of EF-Ts in which Met-19
and Met-20 have been mutated to glutamic acid. The binding constant
between EF-Tu and the M19AM20A mutant of EF-Ts is reduced about 7-fold
compared with the wild-type EF-Ts (Table I). No interaction can be
detected between EF-Tu and the M10EM20E derivative. This observation is not surprising since the presence of Glu in these positions would completely disrupt the hydrophobic core formed by the interaction of
the N-terminal domain of EF-Ts with domain I of EF-Tu. The M19AM20A
derivative shows about a 3-fold lower activity in promoting guanine
nucleotide exchange with EF-Tu and is about 2-fold less active in
stimulating the activity of this factor in polymerization (Fig. 2). As
expected, the M19EM20E mutant is completely inactive in these assays.
Overall, these observations suggest that Met-19 and Met-20 play a
modest role in facilitating the binding of EF-Ts to EF-Tu. Mutation of
these residues to Ala creates two small holes in this portion of the
interface between EF-Tu and EF-Ts. Loss of some of the hydrophobic
binding energy provided by these residues does not have a profound
effect on the interaction between these two proteins. However, it is
clear that hydrophilic groups cannot be tolerated at this portion of
the interface.
The final residue in the N-terminal domain of EF-Ts making contact with
EF-Tu is Lys-23. This residue forms an electrostatic contact with
Asp-141 and contributes to the hydrophilic edge defining the surface of
this region of contact (Fig. 1A). Lys or occasionally Arg
are found at this position in the EF-Ts found in various organisms. When a K23A derivative of EF-Ts is purified from E. coli, it
co-purifies with about the same amount of EF-Tu observed in
preparations of the wild-type factor (data not shown). This observation
suggests that the K23A derivative of EF-Ts binds to EF-Tu essentially
as well as does the wild-type factor. This observation was confirmed by
measuring the binding constant of this mutated derivative which is
indistinguishable from that of the wild-type factor (Table I). The K23A
mutant promotes guanine nucleotide exchange with EF-Tu as effectively
as the normal factor and is only about 2-fold less active in
stimulating the activity of EF-Tu in polymerization (Fig. 2). These
observations indicate that, despite the conservation of a basic residue
at position 23, the electrostatic contacts here contribute little to
the binding between EF-Tu and EF-Ts.
Mutational Analysis of Residues Involved in the Interactions
between Subdomain N of the Core of EF-Ts and Domain 1 of
EF-Tu--
Subdomain N of the core of EF-Ts makes several potentially
important contacts with domain I of E. coli EF-Tu (Fig.
1B). The side chains of Asp-80 and Phe-81 are inserted into
domain I of EF-Tu near the site where the Mg2+ ion
coordinated to GDP is normally located. Asp-80 and Phe-81 are conserved
among all of the EF-Ts sequences determined to date suggesting that
they play an important role in the activity of this protein. These two
residues were mutated in a previous study (10). The D80A and F81A
mutants are 2- to 3-fold less active than wild-type EF-Ts in promoting
guanine nucleotide exchange. The double mutant D81AF81A is about
10-fold less active. We have now examined the contributions of these
two residues to the strength of the interaction between EF-Ts and
EF-Tu. As indicated in Table I, the D80A derivative shows only a
slightly lower (about 2-fold) binding constant for interaction with
EF-Tu. The F81A mutant binds EF-Tu about 6-fold less tightly. The
effects of mutation of both these residues are cumulative, and the
double mutant (D80AF81A) shows about a 12-fold reduction in the binding
constant for EF-Tu (Table I). These values are consistent with the
relative abilities of the mutated derivatives to stimulate the activity
of EF-Tu.
An additional side chain in subdomain N of the core of EF-Ts (Lys-51)
makes an electrostatic contact with Asp-21 in domain I of EF-Tu. This
residue has not been as highly conserved as many others and is actually
a Leu in the mitochondrial factors that are active in promoting guanine
nucleotide exchange with E. coli EF-Tu (18, 19). Hence, it
is unlikely that Lys-51 plays an essential role in stabilizing the
interaction of EF-Tu and EF-Ts. It was not mutated in the present
study.
Two contacts are made between backbone atoms in subdomain N of EF-Ts
and side chains in domain I of EF-Tu. A hydrogen bond is found between
the carbonyl oxygen of Ile-125 and the amine function of Gln-114. An
additional hydrogen bond is found between the carbonyl oxygen of
Gly-126 and an imidazole nitrogen in His-19 (Fig. 1B). To
examine the roles of these contacts in the interaction between EF-Tu
and EF-Ts, two E. coli EF-Tu mutants (H19A and Q114A) were
created. The H19A derivative of EF-Tu has a binding constant for GDP
similar to that of wild-type EF-Tu. This derivative shows a small
reduction (about 3-fold) in its binding constant for wild-type EF-Ts
(Table II). Wild-type E. coli
EF-Ts is about 3-fold less active in stimulating GDP exchange with the
H19A variant of EF-Tu (Fig.
3A). These observations
indicate that contact between the backbone carbonyl of residue Gly-126
in EF-Ts with the side chain of His-19 plays only a minor role in
stabilizing the interaction between EF-Tu and EF-Ts.

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Fig. 3.
Stimulation of GDP exchange with wild-type or
mutated derivatives of E. coli EF-Tu by wild-type
EF-Ts. The stimulation of GDP exchange with EF-Tu was examined as
described under "Materials and Methods." Reaction mixtures
contained the indicated amounts of wild-type EF-Ts and about 80 pmol of
active EF-Tu or the H19A variant (A) or the Q114A variant
(B). Blanks representing the amount of GDP exchange carried
out by EF-Tu alone during this incubation period (about 7 pmol) have
been subtracted from each value.
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To assess the role of the contact between the backbone of Gly-126 of
EF-Ts and Gln-114 of EF-Tu, a Q114A derivative of EF-Tu was prepared.
This derivative has a somewhat weaker binding constant for GDP than the
wild-type factor (about 8 × 107
M
1 compared with 3.3 × 108
M
1, data not shown). It also has a weaker
binding constant for interaction with EF-Ts (about 0.95 × 108, Table II). The reductions in the affinities of this
factor for GDP and EF-Ts offset each other to a significant extent.
Thus, when wild-type E. coli EF-Ts is tested for its ability
to stimulate GDP exchange with the Q114A derivative of EF-Tu, it is
only 2-fold less active than with wild-type EF-Tu (Fig. 3B).
These data indicate that the contact between Gln-114 in EF-Tu and
subdomain N of EF-Ts is only modestly important in the interaction
between these two proteins.
Mutational Analysis of Residues Involved in the Interactions
between Subdomain C of EF-Ts and Domain III of EF-Tu--
Subdomain C
of the core of EF-Ts makes a number of contacts with domain III of
EF-Tu (Fig. 1C). These include His-147, Ile-151, Lys-166,
His-167, Met-170, Ala-174, Val-234, and Met-235. No analysis of the
roles of Ile-151, Met-170, or Ala-174 was carried out in the present
work since these residues are not as highly conserved as many others in
EF-Ts.
His-147 of EF-Ts interacts with Asp-354 through an ion pair and makes a
van der Waals contact with Met-351 in domain III of EF-Tu. His-147 in
EF-Ts was mutated to Ala, and the mutated protein was expressed and
purified from E. coli in buffers lacking GDP. Under these
conditions a small amount of EF-Tu co-purifies with wild-type EF-Ts. In
contrast, no EF-Tu is detected in preparations of the H147A derivative
of EF-Ts. This observation suggests that His-147 may play an important
role in the interaction between these two proteins. This idea was
confirmed when attempts were made to measure the binding constant for
the interaction of the H147A derivative with EF-Tu. The interaction is
too weak to be detected suggesting that there is at least a 100-fold
decrease in the affinity between these two proteins. A small amount of activity in promoting GDP exchange and in stimulating
poly(U)-dependent polymerization can be detected when the
H147A derivative is tested (Fig. 4). This
observation suggests that this derivative of EF-Ts does indeed have
some activity but that its interaction with EF-Tu is significantly
impaired. To assess whether the reduced activity observed is due to the
failure of most of the molecules in the preparation to fold properly,
the CD spectra of this derivative was obtained from 178 to 260 nm and
compared with that of the wild-type factor. No differences in these
spectra were apparent suggesting that the H147A variant folds like the
normal protein (data not shown).

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Fig. 4.
Stimulation of the activities of E. coli EF-Tu by EF-Ts and derivatives mutated in subdomain C. A, the stimulation of GDP exchange with EF-Tu was examined
as described under "Materials and Methods." Reaction mixtures
contained 7.3 µg of expressed EF-Tu (about 80 pmol of active factor)
and the indicated amounts of EF-Ts or its variants. Blanks representing
the amount of GDP exchange carried out by EF-Tu alone (about 7 pmol)
have been subtracted from each value. No GDP binding could be detected
in the absence of EF-Tu indicating that the preparations of EF-Ts used
here were free of EF-Tu. B, stimulation of the activity of
E. coli EF-Tu in poly(U)-directed polymerization by EF-Ts
and its mutants. Reaction mixtures contained 1 pmol of expressed EF-Tu
and the indicated amounts of EF-Ts. Blanks representing the amount of
polymerization catalyzed by EF-Tu alone (about 6 pmol) have been
subtracted from each value.
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Val-234 and Met-235 of EF-Ts form part of a hydrophobic core in the
interaction between EF-Ts and domain III of EF-Tu (Fig. 1C).
These residues were mutated to Ala, and the double mutant was expressed
and purified from E. coli. Significant amounts of EF-Tu
co-purify with this variant of EF-Ts suggesting that it binds well. The
binding constant between EF-Tu and the V234AM235A derivative is reduced
about 3-fold compared with that of the wild-type EF-Ts (Table I). This
derivative is also quite active in stimulating GDP exchange with EF-Tu
and in promoting the activity of this factor in polymerization (Fig.
4). The replacement of Val and Met residues with Ala would be expected
to cause a reduction in the strength of the hydrophobic interactions in
this region of the interface between EF-Tu and EF-Ts. However,
replacement of these residues with charged residues would be expected
to cause a complete disruption of the interaction. This idea was tested by creating a V234EM235E variant. No detectable binding between it and
EF-Tu can be detected (Table I). Finally, the V234EM235E derivative has
no activity in stimulating GDP exchange or polymerization with EF-Tu.
Taken together, these data argue that a hydrophobic interaction in this
portion of the interface is important for allowing the close
association of EF-Tu and EF-Ts, but the exact nature of the residues
forming this interface is not crucial.
The final region of contact tested between subdomain C of EF-Ts and
domain III of EF-Tu is provided by Lys-166 and His-167 in EF-Ts (Fig.
1C). Lys-166 forms an ion pair with Glu-348 of EF-Tu.
His-167 makes contact with this same residue through a bridging water
molecule. Lys-166 is generally a basic residue in the EF-Ts from
prokaryotic organisms but is not conserved in the mitochondrial
factors. His-167 is not a highly conserved residue. When a H166AK167A
variant is purified from E. coli, less EF-Tu is observed
co-purifying with the mutated protein than with the wild-type EF-Ts.
Measurement of the binding constant between the H166AK167A variant and
EF-Tu indicates that this derivative has about a 10-fold lower binding
affinity for EF-Tu than the normal factor. This variant is 2-4-fold
less active in promoting GDP exchange and polymerization with EF-Tu
(Fig. 4). These observations indicate that one or both of these
residues play a modest role in the interaction observed.
To examine the role of these residues more completely, a complementary
derivative of EF-Tu was prepared. This derivative has a mutation that
converts Glu-348 in domain III to Ala. The E348A mutant of EF-Tu was
purified from E. coli. This variant appears to be normal and
binds GDP with approximately the same affinity as the wild-type factor
(data not shown). Surprisingly, when the E348A mutant of EF-Tu is
tested for its ability to bind EF-Ts, it actually has a somewhat higher
binding constant than that observed with the wild-type factor (Table
II). Wild-type EF-Ts can stimulate guanine nucleotide exchange with the
E348A variant as well as with the normal EF-Tu (Fig.
5). This observation seems to be at odds
with the reduction in binding constant in the H166AK167A variant of
EF-Ts. One possible explanation for this difference may be a reflection
of the role of hydrophilic residues at the interfaces of interacting
proteins. These residues are often clustered around the outer rim of
the interacting surface and serve to protect a series of hydrophobic
residues in the core of the interacting surface from water. His-166 and
Lys-167 in EF-Ts may serve this purpose. When both of these residues
are mutated to Ala, a hole in the interacting surface may be formed
that allows the entry of water molecules. The penetration of water may
be sufficient to disrupt the interaction of the hydrophobic residues
forming the core of the surface contacts between subdomain C of EF-Ts and domain III of EF-Tu. Mutation of the single residue Glu-348 on
EF-Tu may not allow water to enter the hydrophobic core since the side
chains of His-166 and Lys-167 may effectively prevent entry of water to
this region. Thus, in this interpretation, His-166 and Lys-167
stabilize the interaction of EF-Tu and EF-Ts not through direct
contacts but indirectly by protecting the hydrophobic interactions occurring in adjacent parts of the interacting surfaces.

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Fig. 5.
Stimulation of GDP exchange by wild-type
EF-Ts using E. coli EF-Tu or its E348A variant. The
stimulation of GDP exchange with EF-Tu was examined as described under
"Materials and Methods." Reaction mixtures contained about 80 pmol
of active EF-Tu and the indicated amounts of EF-Ts. Blanks representing
the amount of GDP exchange carried out by EF-Tu alone during this
incubation period (about 7 pmol) have been subtracted from each
value.
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Deletion Analysis of
-Helix 13 of EF-Ts--
In the crystal
structure of the E. coli EF-Tu·Ts complex, the C-terminal
-helix of EF-Ts folds back across domain I of EF-Tu. This final
helical segment is not present in the EF-Ts found in many species
including the mitochondrial factors (18). Since mitochondrial EF-Ts can
bind E. coli EF-Tu and stimulate the activity of this
factor, it was of interest to test the importance of helix 13 in the
interaction of E. coli EF-Tu and EF-Ts. A derivative of
E. coli EF-Ts (h13-del) was prepared that carries a deletion of the final helix. The binding constant of the deletion derivative to
EF-Tu is only about 2-fold lower than the binding constant of wild-type
EF-Ts (Table I). The activity of EF-Ts in which helix 13 has been
deleted in promoting GDP exchange with E. coli EF-Tu is
about the same as that of wild-type EF-Ts (Fig. 4A). The
activity of the deleted derivative in stimulating the activity of EF-Tu
in polymerization is only slightly lower than that of wild-type EF-Ts
(Fig. 4B). These observations indicate that the final
helical segment in E. coli EF-Ts does not play a crucial role in the interaction of this factor with EF-Tu. This observation is
compatible with the absence of this sequence in many of the EF-Ts
sequences that have been examined to date.
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DISCUSSION |
The binding constants governing the interactions of the mutated
variants of E. coli EF-Ts with EF-Tu indicate that the
contributions of specific residues on the interface to the binding are
very different. Whereas residues such as Lys-9/Arg-12, Met-19/Met-20, Phe-81, His-147, and Lys-166/His-167 are important for the interaction, other residues including Lys-23, Asp-80, and Val-234/Met-235 and helix
13 are not particularly important. More essential contacts appear to
occur between the N-terminal domain and subdomain N of EF-Ts with
domain I of EF-Tu than occur between subdomain C of EF-Ts and domain
III of EF-Tu. Of the residues examined here, Arg-12 and His-147 appear
to be the most crucial for the interaction between EF-Tu and EF-Ts.
The structure of the E. coli EF-Tu·Ts complex suggested
that the mechanism of guanine nucleotide exchange catalyzed by EF-Ts could be explained by the insertion of the side chains of Asp-80 and
Phe-81 near the Mg2+ ion binding site in domain I of EF-Tu
(9). The loss of the Mg2+ ion which is essential for
guanine nucleotide binding would cause the subsequent dissociation of
the GDP moiety. However, mutation of these residues individually has
only modest effects on the activity of EF-Ts (10). The data reported
here indicate that these effects are directly correlated with the
moderate decreases in the affinities of these mutated forms for EF-Tu.
Analysis of the relationship between the abilities of the mutated forms
of EF-Ts to bind EF-Tu and the activities of these derivatives in promoting guanine nucleotide exchange indicates that there is a strong
correlation between these values. A semilog plot of
Kobs versus activity in guanine
nucleotide exchange provides nearly a linear relationship when contacts
between domain I of EF-Tu and EF-Ts are considered (Fig.
6). Changes in the contacts between
domain III of EF-Tu and EF-Ts appear to be somewhat less serious. The correlation between the binding constant and the nucleotide exchange activity suggests that it may not be reasonable to classify certain residues in EF-Ts as being responsible for catalysis and other residues
as being primarily involved in the binding interaction. Rather, the
interaction of EF-Ts with EF-Tu is a more global event in which
multiple small conformational changes arising from the interaction
result in a significant cumulative rearrangement of the guanine
nucleotide binding domain of EF-Tu promoting GDP release.

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Fig. 6.
Relationship between the apparent binding
constant for the interaction between EF-Tu and mutated derivatives of
EF-Ts and the activity of EF-Ts in promoting guanine nucleotide
exchange with EF-Tu. Wild-type EF-Ts ( ), mutants in EF-Ts
contacting domain I of EF-Tu ( ), and mutants in EF-Ts that contact
domain III of EF-Tu ( ). The dashed line shown for the
mutants interacting with domain III should not be taken literally since
only two values were available.
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Examination of the crystal structures of the EF-Tu·Ts complex and
EF-Tu·GDP indicates that one function of EF-Ts is to increase the
separation between domains I and III of EF-Tu (20). This conformational
alteration requires interactions of the N-terminal domain and subdomain
N of the core of EF-Ts with domain I and EF-Tu and the interaction of
subdomain C of EF-Ts with domain III of EF-Tu. The significant
structural rearrangement occurring upon these interactions alters the
conformations of a number of loops in domain I that participate in
nucleotide binding. Thus, a widespread conformational change propagated
through domains I and III of EF-Tu results in GDP release. In this
view, any alteration that affects the ability of EF-Ts to interact with
EF-Tu will have a subsequent effect on nucleotide release. In the
studies reported here, no mutated derivatives of EF-Ts were observed
that could bind EF-Tu but that were unable to catalyze GDP exchange. Thus, no single residue or cluster of residues in EF-Ts can be thought
of as being responsible for the nucleotide exchange reaction. Residues
in this factor important in binding EF-Tu are also important in the
nucleotide exchange reaction. A similar situation is likely to be found
in the variety of nucleotide exchange factors found in the GTPase
superfamily of proteins.
We thank Dr. T. Kawashima for providing the
coordinates of the E. coli EF-Tu·Ts complex.