(Received for publication, May 12, 1997)
From the Department of Chemistry and Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Bovine mitochondrial elongation factor Ts (EF-Tsmt) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu). In contrast, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. EF-Tsmt forms a tight complex with E. coli EF-Tu governed by an association constant of 8.6 × 1010. This value is 100-fold stronger than the binding constant for the formation of the E. coli EF-Tu·Ts complex. To test which domain of EF-Tsmt is important for its strong binding with EF-Tu, chimeras were made between E. coli EF-Ts and EF-Tsmt. Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt increases its binding to E. coli EF-Tu 2-3-fold. Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E. coli EF-Ts decreases its binding to E. coli EF-Tu ~4-5-fold. A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Tsmt binds to E. coli EF-Tu as strongly as EF-Tsmt. A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EF-Tsmt binds E. coli EF-Tu ~25-fold more tightly than E. coli EF-Ts. Thus, the higher strength of the interaction between EF-Tsmt and EF-Tu can be localized primarily to a single subdomain.
The classical model for the elongation cycle of protein biosynthesis was developed on observations made with Escherichia coli EF-Tu.1 EF-Tu forms a ternary complex with aminoacyl-tRNA and GTP that promotes the binding of the aminoacyl-tRNA with the A-site of the ribosome. EF-Tu then hydrolyzes the bound GTP and is released from the ribosome as an EF-Tu·GDP complex (1). A second elongation factor (EF-Ts) promotes the release of GDP, forming an intermediate EF-Tu·Ts complex (2). In E. coli, GTP binds to the EF-Tu·Ts complex, promoting the release of EF-Ts and the formation of an EF-Tu·GTP complex. A new ternary complex can then form, and the cycle repeats. In Thermus thermophilus, a dimeric complex (EF-Tu·Ts)2 occurs through the interaction of two EF-Tu molecules with a stable EF-Ts dimer (3). In contrast to the E. coli EF-Tu·Ts complex, the T. thermophilus complex is not dissociated to a significant extent by either GDP or GTP alone (4).
Mitochondrial EF-Tu and EF-Ts have been purified from bovine liver as a tightly associated complex (EF-Tu·Tsmt) (5, 6). The EF-Tu·Tsmt complex differs from the corresponding E. coli complex in that EF-Tu·Tsmt is not readily dissociated by guanine nucleotides (5, 6). Furthermore, no significant amounts of intermediates equivalent to EF-Tu·GTP or EF-Tu·GDP can be detected in the animal mitochondrial system. However, mammalian EF-Tu·Tsmt forms a ternary complex with GTP and aminoacyl-tRNA (7). The basic steps of the bacterial elongation cycle thus appear to be occurring in mammalian mitochondria. However, the equilibrium constants that govern the interaction of EF-Tu with EF-Ts and guanine nucleotides appear to be significantly different.
The cDNAs encoding EF-Tumt and EF-Tsmt have been cloned and sequenced from bovine and human liver (8, 9). Sequence analysis indicates that EF-Tumt has 56% identity to E. coli EF-Tu while EF-Tsmt is less than 30% identical to E. coli EF-Ts. When EF-Tsmt is expressed and purified from E. coli, it forms a 1:1 complex with E. coli EF-Tu (EF-TuEco·Tsmt) (9). This heterologous complex is very resistant to dissociation by guanine nucleotides even at high concentrations of GDP or GTP (10). This feature of the heterologous complex is quite reminiscent of the native EF-Tu·Tsmt complex. Thus, it is apparently the nature of EF-Ts that determines the strength of its interaction with EF-Tu. However, it is not clear what features of EF-Tsmt modulate its tight interaction with EF-Tu.
The crystal structures of trypsin-modified E. coli
EF-Tu·GDP and of Thermus aquaticus EF-Tu
complexed with a nonhydrolyzable GTP analogue have been
determined (11, 12). Analysis of these structures indicates
that EF-Tu folds into three domains. Domain I encompasses
about the first 200 residues and contains the guanine nucleotide binding site. Domains II and III are each ~100
residues long. All three domains are involved in binding
aminoacyl-tRNA (13). The structure of the E. coli
EF-Tu·Ts complex has also been determined (Fig.
1) (14). This structure
indicates that EF-Ts consists of 4 structural modules: 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 further divided into Subdomain N (residues 55-140) and
Subdomain C (residues 141-179 and 229-263). The N-terminal domain,
Subdomain N, and the C-terminal module interact with Domain I of EF-Tu,
whereas Subdomain C interacts with Domain III of EF-Tu.
EF-Tsmt binds to EF-Tu more tightly than does E. coli EF-Ts. This work attempts to determine the regions of the
EF-Tsmt giving rise to its higher affinity for EF-Tu.
Clones encoding
His-tagged variants of E. coli EF-Ts and EF-Tsmt
in pET24C(+) were prepared previously (9, 15). To make a mutant of
E. coli EF-Ts lacking the N-terminal domain (residues 1 to
53), a fragment of the E. coli EF-Ts gene containing
sequences encoding amino acid residues 54-282 was amplified by
polymerase chain reaction (PCR) using primer ETsP4
(ccggctcgagagactgcttggacatcgcagc) and ETsN
(cgggatcccatatgaacgttgctgctgacggc). The PCR fragment was digested with
NdeI and XhoI and cloned into NdeI-
and XhoI-digested pET24C(+), giving the construct pETs
N.
To make a mutated form of EF-Tsmt lacking the N-terminal
domain (residues 1-56), a fragment of EF-Tsmt gene
containing the sequence encoding amino acid residues 57-283 was
amplified by PCR using primer BMC-N (cgggatcccatatgcgtaagaccaaagaaggt) and C-XhoI (ccgctcgagttcggcgtctgctgcgtc). The PCR fragment
was digested with NdeI and XhoI and cloned into
NdeI- and XhoI-digested pET24C(+), giving the
construct pTsmt
N.
To make Chimeras I and II (see Fig. 5), a
HindIII site was introduced into the EF-Tsmt
gene at nucleotide sequence position 342 by site-directed mutagenesis
using the ChameleonTM Double-Stranded, Site-Directed
Mutagenesis Kit (Stratagene). Primer MP15
(cttcctcccatgaagcttagcagctttact) was used for the mutagenesis. The HindIII site is underlined and
the mutated residues are shown in boldface. This mutation changes residue Arg-54 to Lys-54. In the human EF-Tsmt gene, the
amino acid residue at the same position is Lys-54. The N-terminal
domain of E. coli EF-Ts from the N terminus to residue 49 was amplified by PCR using primer ETsP1
(gggaagcttcatatggctgaaattaccgcatccct) and ETsP2 (cccaagcttcgctgctttaatagcaccg). The
E. coli EF-Ts sequence is underlined and the restriction
enzyme sites are boldface. To make Chimera I, the PCR fragment was
digested with NdeI and HindIII and used to
replace the NdeI to HindIII fragment of
EF-Tsmt using standard methods. To make Chimera II, a PCR
fragment of E. coli EF-Ts encompassing amino acid residues
52 to the C terminus was amplified using primer ETsP3
(cccaagcttgcaggcaacgttgctgctgac) and ETsP4
(ccggctcgagagactgcttggacatcgcagc). The E. coli EF-Ts sequence is underlined and the restriction enzyme sites are boldface. This PCR fragment was digested with HindIII
and XhoI and used to replace the HindIII to
XhoI fragment of EF-Tsmt gene.
Chimeras III and IV were prepared using chimeric primers and two rounds
of PCR. This strategy, which is outlined in
Fig. 2, permits the preparation of
chimeric proteins at any position without the need to introduce new
restriction sites. To make Chimera III, a portion of
EF-Tsmt gene from the N terminus to amino acid residue 162 in the mature form of this factor (9) was amplified by PCR using primer
Vec-2 (taggggaattgtgagcggataac), derived from the pET24C(+) vector, and
primer MP25 (agaacccagaacgtcgccgctggcaccttcac). This primer is in itself a chimeric piece of DNA. The sequence shown in
boldface is derived from the EF-Tsmt gene, while the underlined sequence is from the E. coli EF-Ts gene. DNA was
amplified from 100 ng of plasmid DNA carrying the EF-Tsmt
gene in a 100-µl reaction mixture containing 100 pmol of each primer
and 2.5 units of Taq polymerase. PCR was carried out for 20 cycles (94 °C for 1 min, 55 °C for 2 min, and 72 °C for 2 min). The DNA fragment from this amplification reaction was purified by
gel electrophoresis. A chimeric EF-Ts fragment was then amplified by
using primer ETsP4 and the PCR fragment prepared above as primers with
the E. coli EF-Ts gene in pET24C(+) as the template and the
PCR conditions described above. This second DNA fragment was purified
by gel electrophoresis, digested with NdeI and
XhoI, and cloned into pET24C(+) to make Chimera III.
To make Chimera IV, a PCR fragment of the E. coli EF-Ts gene in pET24C from the N terminus to amino acid residue 148 was amplified by primer Vec-2 and MP24 (agagccaacatagaaaccttccagcgcagcaac). This primer is a chimera in which the underlined sequence is derived from the E. coli EF-Ts gene while the sequence in boldface is derived from the EF-Tsmt gene. The PCR conditions described above were used, and the amplified DNA was purified by gel electrophoresis. This PCR fragment and primer C-XhoI were then used as primers to amplify a chimeric EF-Ts fragment using the EF-Tsmt DNA as template and the conditions described above. This second PCR fragment was purified by gel electrophoresis, digested with NdeI and XhoI, and cloned into pET24C(+) to make a construct encoding Chimera IV. To make Chimera V, Chimeras II and III were both digested with PstI and XhoI. The 0.7-kilobase fragment of Chimera III was ligated to the 5.5-kilobase fragment of Chimera II.
Expression and Purification of EF-Tu and EF-TsThe His-tagged forms of E. coli EF-Tu and EF-Tumt were expressed and purified as described previously (15). E. coli EF-Ts, Chimera I, and Chimera IV were expressed as described previously (15) and purified under two conditions. In the first set of conditions, 10 mM Mg2+ was included in the buffers. In the second set of conditions, 10 µM GDP was also included in the preparation buffers. Expression of EF-Tsmt and Chimeras II, III, and V was carried out as described (9), and these proteins were purified under native and denaturing conditions. Under native conditions, 10 mM Mg2+ was included in the buffers but no GDP was added. When cell extracts were prepared under native conditions, EF-Tsmt was isolated as 1:1 complex with E. coli EF-Tu. To purify EF-Tsmt, Chimera II, and Chimera III free of E. coli EF-Tu, the EF-TuEco·Tsmt complexes were denatured. The His-tagged forms of EF-Tsmt and the chimeras were purified through a nickel-nitrilotriacetic acid column and renatured (10). The protein concentrations were determined by the Micro-Bradford method (Bio-Rad). Samples (~10 µg each) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie Blue (16).
AssaysThe activities of E. coli EF-Ts, EF-Tsmt, and their chimeras were determined by measuring their abilities to promote guanine nucleotide exchange with E. coli EF-Tu·GDP and to stimulate the activity of E. coli EF-Tu in poly(U)-directed polymerization on E. coli ribosomes (17, 18). The activities of E. coli EF-Ts, EF-Tsmt, and their chimeras were also determined by their ability to stimulate the activity of expressed EF-Tumt in the poly(U)-directed polymerization of phenylalanine on E. coli ribosomes (10).
Binding Constant MeasurementsTo determine the binding constants of EF-Tsmt, Chimeras III and V to E. coli EF-Tu, 1:1 complexes of EF-TuEco·Tsmt, EF-TuEco·Chimera III, or EF-TuEco·Chimera V (0.2 to 0.4 µM) were incubated in 500-µl reactions containing 10 µM or 20 µM [3H]GDP (200 cpm/pmol) in Buffer A (50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 1 mM dithiothreitol, and 0.1 M NH4Cl) at 0 °C for 90 min. The amount of EF-Tu·GDP present at equilibrium was determined using a nitrocellulose filter binding assay (17). The amount of active EF-Tu in the complexes was estimated to be ~50% based on its ability to bind GDP. To determine the binding constants of E. coli EF-Ts, Chimera I and Chimera II to E. coli EF-Tu, EF-Tu·[3H]GDP (0.5 µM, 50 pmol), [3H]GDP (500 cpm/pmol) (5 or 10 µM) and EF-Ts or its chimeras (1-3 µM, 100-300 pmol) were incubated in a reaction mixture (100 µl) at 20 °C for 30 min in the buffer indicated above. The amount of EF-Tu·GDP at equilibrium was measured by the nitrocellulose filter binding assay (17). Kobs for the reaction
![]() |
(Eq. 1) |
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![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
When mitochondrial EF-Tsmt is expressed in E. coli as a His-tagged protein, it forms a 1:1 complex with E. coli EF-Tu (9). Free EF-Tsmt can be purified by denaturing this complex and then allowing renaturation of the EF-Tsmt (10). E. coli EF-Ts has also been overexpressed in E. coli and purified as a His-tagged protein (15). When no GDP is added to the isolation buffers, a small amount of E. coli EF-Tu co-purifies with the E. coli EF-Ts following chromatography on nickel-nitrilotriacetic acid resins. E. coli EF-Ts was prepared free of E. coli EF-Tu by using buffers containing GDP and Mg2+.
The abilities of EF-Tsmt and E. coli EF-Ts to
stimulate GDP exchange and poly(U)-directed polymerization with
E. coli EF-Tu were tested
(Fig. 3, A and B).
The purified EF-Tsmt stimulates guanine nucleotide exchange
with E. coli EF-Tu and also stimulates the poly(U)-directed
polymerization of phenylalanine (Fig. 3A). However,
substantially higher levels of EF-Tsmt are required to achieve the same degree of stimulation observed with E. coli
EF-Ts. Nearly 10-fold higher concentrations of EF-Tsmt are
required to promote the same amount of nucleotide exchange obtained
with E. coli EF-Ts (Fig. 3A). The activity of
EF-Tsmt in stimulating poly(U)-directed polymerization with
E. coli EF-Tu is ~25% of that obtained with E. coli EF-Ts (Fig. 3B).
EF-Tsmt stimulates the activities of both EF-Tumt and E. coli EF-Tu in poly(U)-directed polymerization (Fig. 3, B and C). E. coli EF-Ts cannot stimulate the activity of EF-Tumt (Fig. 3C). This observation indicates that E. coli EF-Ts may be unable to bind EF-Tumt or that it binds to EF-Tumt much more weakly than GDP does. Alignment of the primary sequence of E. coli EF-Tu and EF-Tumt indicates that these two factors are 56% identical. In addition, all of the residues in E. coli EF-Tu that are in contact with EF-Ts in the crystal structure are identical or are conservative replacements in EF-Tumt. Thus, the failure of E. coli EF-Ts to stimulate the mitochondrial factor in translation is surprising.
As indicated above, higher levels of EF-Tsmt are required to produce the same degree of stimulation of E. coli EF-Tu observed with low levels of E. coli EF-Ts. Previous results have suggested that EF-Tsmt binds to E. coli EF-Tu more tightly than does E. coli EF-Ts (10). This idea is based on the observation that the heterologous complex EF-TuEco·Tsmt is not readily dissociated by guanine nucleotides while the homologous E. coli complex is. Thus, the lower activity of EF-Tsmt probably arises from its slow release from EF-Tu which reduces the rate of ternary complex formation. To test this idea, we have determined the approximate equilibrium association constant for the binding of EF-Tsmt to E. coli EF-Tu and have compared this value with that obtained with the homologous factor.
To determine the association constant for the binding of EF-Tsmt to E. coli EF-Tu, the Kobs of the following reaction was measured as described under "Materials and Methods."
![]() |
(Eq. 4) |
|
Analysis of the protease sensitivity of EF-Ts (20) and the x-ray structure of the E. coli EF-Tu·Ts complex (14) shows that the N-terminal region of E. coli EF-Ts folds into an independent domain (Fig. 1). This region is essential for the ability of E. coli EF-Ts to stimulate guanine nucleotide exchange with EF-Tu (20). Sequence alignment indicates that there is significant homology between E. coli EF-Ts and EF-Tsmt in the N-terminal domain and it is likely that it will fold in a similar three-dimensional structure. It was, therefore, of interest to determine whether the N-terminal domain of EF-Tsmt was also important for its binding to EF-Tu or whether other interactions of this factor could compensate for the loss of this domain. To examine this question, N-terminal deletion mutants of E. coli EF-Ts and EF-Tsmt were constructed (Fig. 5). The EF-Ts deletion mutants were tested for their abilities to bind E. coli EF-Tu and to stimulate the activities of E. coli EF-Tu and EF-Tumt in polymerization. The N-terminal deletion mutant of E. coli EF-Ts is unable to stimulate the activity of EF-Tu in guanine nucleotide exchange or in poly(U)-directed polymerization (data not shown). This observation is in agreement with previous results showing that a proteolytic derivative of E. coli EF-Ts lacking the N-terminal domain is unable to stimulate the activity of EF-Tu (20). The N-terminal deletion mutant of EF-Tsmt is unable to bind E. coli EF-Tu (data not shown). It is also inactive in stimulating the activity of either E. coli EF-Tu or EF-Tumt (data not shown). These data indicate that the N-terminal domain of EF-Tsmt, like that of E. coli EF-Ts, is important for its function in protein synthesis. These data, while indicating that the N-terminal domain is important for the interaction of EF-Tsmt with EF-Tu, do not provide any insight into whether this region plays a role in the stronger affinity for EF-Tu observed with EF-Tsmt.
Predicted Secondary Structure of EF-Tsmt and Analysis of Chimeric Proteins between E. coli and Mitochondrial EF-TsIn
the crystal structure of the E. coli EF-Tu·Ts complex
(Fig. 1) (14), the N-terminal domain and Subdomain N of the core interact with Domain I of E. coli EF-Tu. Subdomain C of the
core interacts with Domain III of E. coli EF-Tu. As
indicated above, EF-Tsmt binds to E. coli EF-Tu
more tightly than does E. coli EF-Ts. It has not yet been
possible to make direct measurements of the binding constant of
EF-Tsmt for EF-Tumt. However, it is clear that
guanine nucleotides cannot dissociate the EF-Tu·Tsmt complex (6). Thus, it is likely that EF-Tsmt will also have a high affinity for EF-Tumt. To help determine which
region(s) might be important for the strong interaction of
EF-Tsmt with EF-Tu, an analysis of the possible structure
of EF-Tsmt was carried out using the 3-dimensional
structure of E. coli EF-Ts and several secondary structure
prediction programs as a guide (Fig. 4).
The overall lengths of E. coli EF-Ts and the mature form of
EF-Tsmt are the same (9). However, the two factors appear
to have several significant differences in their overall structure.
EF-Tsmt aligns well with E. coli EF-Ts in the
N-terminal domain, and this region of the protein most likely folds
into 3 helices as observed for the E. coli factor. The first
2 strands of -sheet in Subdomain N and helices h4 and h5 are also
predicted to be present in EF-Tsmt. However, Subdomain N is
interrupted by an insertion of ~20 amino acids. The precise position
of this insertion is difficult to predict since the two proteins show
very little primary sequence homology in this region making the
alignment difficult. The remainder of Subdomain N including helices h6
and h7 and the final strand of the
-sheet (s3) are all predicted to
be present. Subdomain C is predicted to begin with
-strand s4 which
is followed by an insertion of ~12 residues before strands s5 and s6
and helices h8 and h12 are found. The dimerization domain (helices h9,
h10, and h11) which is involved in contacts between two EF-Ts molecules in the crystal structure of the EF-Tu·Ts complex is largely missing from EF-Tsmt. Only portions of what may be helix h10 appear
to be present. Finally, the C-terminal module (h13) present in E. coli EF-Ts is missing in EF-Tsmt.
The secondary structural analysis described above provided a good tool
for the analysis of what regions of EF-Tsmt are important for the tight binding to EF-Tu observed with EF-Tsmt. Based
on this analysis, a series of chimeric proteins consisting of portions of E. coli EF-Ts and EF-Tsmt were constructed
(Fig. 5). Chimeras were first constructed
by exchanging the N-terminal domains (h1 through h3) of E. coli EF-Ts and EF-Tsmt (Fig. 5, Chimeras I and II).
These two chimeric proteins were expressed in E. coli as His-tagged proteins and purified by chromatography on
nickel-nitrilotriacetic acid columns. In addition, His-tagged forms of
EF-Tsmt and E. coli EF-Ts were also expressed
and purified. All of these proteins were initially purified from cell
extracts in buffers without GDP but containing Mg2+. The
purified proteins were analyzed by SDS-PAGE followed by Coomassie Blue
staining (Fig. 6). Under these conditions, EF-Tsmt is
purified as a 1:1 complex with E. coli EF-Tu
(Fig. 6, lane 1). In contrast,
E. coli EF-Ts binds to E. coli EF-Tu much less tightly, and only ~1 mol of EF-Tu is present for every 20 mol of
E. coli EF-Ts (Fig. 6, lane 2). The weaker
binding of E. coli EF-Ts prevents it from competing
effectively for EF-Tu with the guanine nucleotides and aminoacyl-tRNA
present in the extract. Chimera I which carries the N-terminal domain
of EF-Tsmt binds E. coli EF-Tu ~2-3-fold
better than E. coli EF-Ts does (Fig. 6, lane 3).
It is isolated with a higher ratio of EF-Tu present in the preparations
(~1:10) than observed with E. coli EF-Ts. This observation
suggests that the N-terminal domain has a small effect on increasing
the affinity of EF-Ts for EF-Tu. Chimera II binds to E. coli
EF-Tu much more tightly than does E. coli EF-Ts but it
somewhat less tightly than EF-Tsmt (Fig. 6, lane
4).
To obtain more quantitative measurements of the affinity of these chimeras for E. coli EF-Tu, equilibrium association constants were determined as described above for the normal proteins. For these experiments, E. coli EF-Ts and Chimera I were prepared free of EF-Tu by the use of buffers containing GDP during the preparation of the factors. EF-Tsmt and Chimera II were prepared by the denaturation of the EF-TuEco·Tsmt complex followed be renaturation of the EF-Tsmt (10). Analysis of these preparations on SDS-PAGE indicated that they were free of EF-Tu (data not shown). As indicated in Table I, replacing the N-terminal domain of E. coli EF-Ts with that of EF-Tsmt (Chimera I) increases the binding constant for EF-Tu ~2-3-fold. This observation suggests that this region of EF-Tsmt has a small effect on the strength of the interaction with EF-Tu. In the complementary construct (Chimera II), replacing the N-terminal domain of EF-Tsmt with that of E. coli EF-Ts decreases the binding constant of EF-Tsmt to E. coli EF-Tu ~4-5-fold. Chimera II which is predominantly derived from EF-Tsmt still binds EF-Tu ~20-fold more tightly than does E. coli EF-Ts. These observations indicate that the strength of the interaction observed with EF-Tsmt is governed primarily by sequences from h4 to the C terminus with a small contribution from sequences in the N-terminal domain.
To localize the region giving EF-Tsmt its stronger affinity
for EF-Tu more closely, two more chimeras were prepared (Fig. 5,
Chimeras III and IV). The preparation of these chimeras was based on
the observation that the N-terminal half (the N-terminal domain and
Subdomain N, h1 through s3) and the C-terminal half (from s4 to the C
terminus) of E. coli EF-Ts fold somewhat independently in
the crystal structure of the E. coli EF-Tu·Ts complex
(Fig. 1). However, the three-stranded -sheet structure in Subdomain N forms an interface with the three-stranded sheet in Subdomain C (Fig.
1) and these chimeras must be able to form this structure to fold
correctly. Chimera III consists of the N-terminal domain and Subdomain
N of EF-Tsmt with the C-terminal half of E. coli EF-Ts while Chimera IV is the reverse construct (Fig. 5). Chimera III
was purified as a 1:1 complex with E. coli EF-Tu (Fig. 6, lane 5). This observation suggests that it has folded
correctly and that the determinants for the strong interaction between
EF-Tsmt and EF-Tu reside in the NH2-terminal
half of the protein. The association constant for the binding of
Chimera III to EF-Tu (Table I) indicated that this chimera binds to
EF-Tu as strongly as does the native EF-Tsmt. This
observation indicates that all of the stronger binding between
EF-Tsmt and EF-Tu arises from the N-terminal domain and
Subdomain N of EF-Tsmt. This region of EF-Ts is in contact
with Domain I of EF-Tu. These data also indicate that the C-terminal
half of EF-Tsmt does not contribute significantly to the
ability of EF-Tsmt to bind EF-Tu more tightly than E. coli EF-Ts.
The reciprocal construct (Chimera IV, Fig. 5) was unable to bind
E. coli EF-Tu (Fig. 6, lane 6) and probably folds
incorrectly. This observation suggests that the interface between
Subdomain N and Subdomain C does not form correctly in this construct.
Chimera IV is expressed well in E. coli and does not appear
to form inclusion bodies or to be readily degraded. Hence, it is
reasonable to suggest that Chimera IV has significant structure but
that certain features crucial to its interaction with EF-Tu are not
correctly positioned. A detailed examination of the -sheet interface
indicates that sequence differences in s6 affect the interaction of s6
with s3, possibly resulting in a distortion of the interface between
the
-sheets.
The data obtained with Chimeras I, II, and III suggest that sequences within Subdomain N of EF-Tsmt are primarily responsible for the stronger affinity of this factor for EF-Tu. To confirm this idea, an additional chimera was prepared (Chimera V, Fig. 5) in which Subdomain N of E. coli EF-Ts was replaced with that of EF-Tsmt. When Chimera V is prepared from E. coli, it co-purifies with considerable amounts of EF-Tu as does EF-Tsmt (Fig. 6, lane 7). This chimera is longer than the others and migrates on SDS-PAGE at a higher molecular weight than the other chimeras, due to the insertion present in Subdomain N of EF-Tsmt. The association constant for the binding of Chimera V to EF-Tu (Table I) was ~25-fold higher than that of E. coli EF-Ts. This observation agrees with the idea that sequences in Subdomain N of EF-Tsmt are primarily responsible for the stronger interaction of this factor with EF-Tu. Since there is an insertion of ~20 amino acids in this region, contacts between one or more of these residues may be occurring between EF-Tsmt and Domain I of EF-Tu.
Stimulation of the Activity of EF-Tu by EF-Ts and Its ChimerasAs indicated in Fig. 3, E. coli EF-Ts is
active with its endogenous factor but not with EF-Tumt. In
contrast, EF-Tsmt can stimulate the activities of both
bacterial and mitochondrial EF-Tu, although it is less efficient than
E. coli EF-Ts in stimulating the activity of E. coli EF-Tu. The activities of the chimeric EF-Ts proteins in
stimulating guanine nucleotide exchange with E. coli EF-Tu
were tested. As indicated in Fig.
7A, replacing the N-terminal domain of E. coli
EF-Ts with that of EF-Tsmt in Chimera I results in a factor
that has the same activity as E. coli EF-Ts in promoting
guanine nucleotide exchange. This level of activity, like that of
E. coli EF-Ts, is ~10-fold higher than the activity
observed with EF-Tsmt. Chimera II, in which the N-terminal domain of E. coli EF-Ts has replaced that in
EF-Tsmt, has about one-half of the activity observed with
E. coli EF-Ts (Fig. 7A). This chimera has
3-4-fold higher activity than that seen with EF-Tsmt. The
activity of Chimera III in stimulating GDP exchange is about the same
as that observed with EF-Tsmt (Fig. 7B). This chimera has the entire N-terminal half of EF-Tsmt and binds
to EF-Tu as tightly as EF-Tsmt. Chimera IV is not active in
stimulating GDP exchange as would be expected from its apparent
inability to bind EF-Tu. The activity of Chimera V is slightly higher
than that of EF-Tsmt but significantly lower than E. coli EF-Ts. Overall, these results indicate that
EF-Tsmt and the chimeras that bind to EF-Tu more tightly
have lower activities in promoting guanine nucleotide exchange.
The activities of the chimeras in stimulating the activity of E. coli EF-Tu in polymerization were also tested (Fig.
8A). In this assay, as in the GDP exchange assay,
EF-Tsmt is less active than E. coli EF-Ts
(Figs. 3 and 8A). The activity
of Chimera I is very similar to that of E. coli EF-Ts. The
activity of Chimera II is only slightly lower than that of E. coli EF-Ts and much higher than that of EF-Tsmt. This
assay is probably somewhat less sensitive to changes in the affinity of
EF-Ts for EF-Tu, since the formation of the EF-Tu·Ts complex is
coupled to the subsequent very favorable formation of the ternary
complex. This coupling might tend to offset the stronger interaction
between EF-Tu and EF-Ts to some extent. The activity of Chimera III is
about the same as that observed with EF-Tsmt. Since this
chimera has the same affinity for EF-Tu as does EF-Tsmt,
this result is to be expected. These data further indicate that
replacing the N-terminal domain of EF-Tsmt (Chimera II)
increases the activity of EF-Tsmt in stimulating the
activity of E. coli EF-Tu, while replacing the C-terminal
half of EF-Tsmt does not appear to affect the activity of
EF-Tsmt.
As shown in Fig. 3C, E. coli EF-Ts is not able to stimulate the activity of EF-Tumt in polymerization. The activities of the chimeras in stimulating EF-Tumt in polymerization were tested (Fig. 8B). Replacing the N-terminal domain of E. coli EF-Ts with the corresponding region of EF-Tsmt (Chimera I) does not restore activity, indicating that the interaction between Domain I of EF-Tu and the N-terminal domain of EF-Ts is not responsible for the lack of activity observed with E. coli EF-Ts. In agreement with this idea is the observation that Chimera II, in which the N-terminal domain of EF-Tsmt is replaced by the corresponding region from E. coli EF-Ts, is quite comparable with that of EF-Tsmt (Fig. 8B). Chimera III, in which the C-terminal half of EF-Tsmt has been replaced by that from E. coli EF-Ts, has very little or no activity in stimulating EF-Tumt in polymerization. Chimera V could not be tested in this assay since it could not be refolded into an active conformation following denaturation of the EF-TuEco·Chimera V complex. These data indicate that E. coli EF-Ts cannot stimulate the activity of EF-Tumt because of a failure of the C-terminal half of this factor to interact correctly with Domain III of EF-Tumt. This observation is surprising since residues in EF-Tu making contact with Subdomain-C of EF-Ts in the crystal structure have been conserved in the mitochondrial factor.
The data presented here show that EF-Tsmt binds to E. coli EF-Tu ~100-fold more tightly than E. coli EF-Ts. Analysis of the chimeric proteins indicates that sequences in Subdomain N of the core of EF-Ts are primarily responsible for the difference in the tightness of binding observed while the N-terminal domain also makes a small contribution. Six residues in the N-terminal domain of E. coli EF-Ts make contact with residues in Domain I of E. coli EF-Tu (14). Some of these residues are conserved in EF-Tsmt while others are primarily conservative replacements. These latter residues in EF-Tsmt may account for the small effect on binding contributed by the N-terminal domain.
In the interactions between Domain I of E. coli EF-Tu and Subdomain N of the core of E. coli EF-Ts, four residues (Asp-80, Phe-81, Ile-125, and Gly-126) of E. coli EF-Ts are directly involved. The only residue in EF-Tsmt that is different in this group is the conservative replacement of Leu-151 for the corresponding residue Ile-125 in E. coli EF-Ts. Since Ile-125 of E. coli EF-Ts makes a backbone contact with EF-Tu, it is unlikely that Leu-151 of EF-Tsmt contributes to the stronger interaction observed between EF-Tsmt and EF-Tu. D80A and F81A mutants of E. coli EF-Ts bind to EF-Tu much more weakly than does wild type EF-Ts. However, the corresponding D84A and F85A mutants of EF-Tsmt still bind to E. coli EF-Tu as tightly as does the wild-type factor (15). These results suggest that Asp-84 and Phe-85 of EF-Tsmt do not contribute to the stronger interaction of EF-Tsmt with E. coli EF-Tu. However, as indicated in Fig. 4, Subdomain N of the core of EF-Tsmt has an insertion of ~20 residues. Although the exact position of this insertion is difficult to assess, it is likely that these residues account for the stronger interaction between EF-Tsmt and EF-Tu.
Decreased Tightness of Binding between EF-Tsmt Increases Its Ability to Stimulate the Activity of EF-TuAlthough EF-Tsmt interacts with E. coli EF-Tu very well, it is significantly less active than E. coli EF-Ts in stimulating the activity of E. coli EF-Tu. One explanation for the lower activity observed is that the strong binding actually inhibits the activity of EF-Tsmt. The reaction of E. coli EF-Ts in stimulating guanine nucleotide exchange can be described as
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(Eq. 5) |
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Despite the 56% identity in sequence between
E. coli EF-Tu and EF-Tumt, E. coli
EF-Ts is unable to stimulate the activity of EF-Tumt in
polymerization. This result suggests that E. coli EF-Ts does
not interact very well with EF-Tumt or that the interaction occurring fails to result in effective nucleotide exchange.
Unfortunately, the poor binding of guanine nucleotides to
EF-Tumt does not allow a direct measure of the exchange
reaction. Since Chimera II is able to stimulate the activity of
EF-Tumt, the N-terminal domain of E. coli EF-Ts
probably interacts with EF-Tumt quite well. The low
activities of Chimeras I and III with EF-Tumt suggests that the C-terminal half of E. coli EF-Ts does not form a good
interaction with Domain III of EF-Tumt. This observation is
unexpected since the residues in EF-Tu making contact with Subdomain C
of EF-Ts in the crystal structure have been conserved in
EF-Tumt. On the other hand, the alignment of the sequences
of EF-Ts from a number of organisms shows that the C-terminal half of
EF-Ts is not as conserved as the N-terminal half. E. coli
EF-Ts has an extra -helix (h13) at the C-terminal terminus compared
with EF-Tsmt. In addition, E. coli
EF-Ts has a dimerization domain that is not present in EF-Tsmt. These structural differences may lead to the
inability of E. coli EF-Ts to stimulate the activity of
EF-Tumt.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L37935.