(Received for publication, July 20, 1995; and in revised form, September 22, 1995)
From the
In a search for active-site residues of the Tsp protease, 20
positions were individually mutated to alanine, the mutant strains were
assayed for growth defects in vivo, and the purified proteins
were assayed for proteolytic activity in vitro. Alanine
substitutions at three positions, Ser-430, Asp-441, and Lys-455, result
in inactive proteases that have structures and substrate-binding
properties similar to wild type, suggesting that the side chains at
these positions participate in catalysis. Replacing Ser-430 with
cysteine results in a partially active protease, which is inhibited by
cysteine-modifying reagents. Replacing Asp-441 with asparagine does not
significantly affect activity. However, other residues, including
histidine and arginine, cannot functionally replace Lys-455. These data
are consistent with a serine-lysine dyad mechanism, similar to those
proposed for the LexA-like proteases, the type I signal peptidases, and
the class A -lactamases.
Tsp is a periplasmic protease of Escherichia
coli(1) , which has been implicated in the processing of
penicillin binding protein 3 (2) and TonB ()and
appears to play some role in fatty acid transport mediated by the FadL
protein(3) . Deletion of the tsp gene (also known as prc) causes a temperature-dependent growth defect under
conditions of osmotic stress(2) . Biochemical studies in
vitro suggest that Tsp selects substrate proteins based on the
identity of side chains and functional groups at their C termini. For
example, amidation of the
-carboxyl group of a good peptide
substrate prevents cleavage by Tsp(4) . Moreover, Tsp cleaves
variants of Arc repressor and the N-terminal domain of
-repressor,
which has apolar C-terminal residues but does not cleave variants of
comparable stability that have polar C-terminal
sequences(1, 4) .
Tsp does not share sequence
homology with well characterized proteases or protease active-site
sequences and cannot be classified as a serine, cysteine, aspartic, or
metallo protease based on inhibitor studies(1) . A portion of
the Tsp sequence is homologous to a sequence motif repeated 4 times in
the interphotoreceptor retinoid binding protein (IRBP) ()of
mammals(1) . It has been proposed that Tsp binds the apolar C
termini of its substrates in the same fashion as the IRBP repeats bind
retinoids. Recently, four bacterial genes encoding sequences with
homology to Tsp have been determined (see Fig. 1). We assume
that many of these genes encode proteases, although this is supported
by genetic or biochemical evidence only in the case of
CtpA(5) .
Figure 1: Sequence homology. Top, schematic representation of the Tsp sequence. Regions of homology with CtpA,, Aqu, BaoCtpA, and Ngu and with the IRBP repeats (1) are shown. Bottom, sequence alignments. Numbers refer to the Tsp sequence, and residues that are identical in all sequences are shaded. Open circles indicate alanine substitutions that result in an active Tsp protease. Squares indicate substitutions that result in inactivity and structural perturbations. Black circles indicate substitutions that result in inactivity without structural perturbations or substrate binding defects. The accession numbers for the sequences are Tsp (P23865), CtpA (L25250), Aqu (S18125), BaoCtpA (L37094), and Ngu (U11547).
In this paper, we construct Tsp mutants by
site-directed mutagenesis, test the mutant strains for growth defects in vivo, and assay the mutant proteins for protease activity in vitro. Inactive mutant proteins are assayed for gross
structural perturbations using circular dichroism spectrometry.
Finally, the binding of substrates to the inactive but structurally
intact proteins is assayed. Using these approaches, we identify three
Tsp residues (Ser-430, Asp-441, and Lys-455), which have properties
expected for active-site residues. The behavior of proteins altered at
these positions suggests that Tsp may use a serine-lysine mechanism
similar to those proposed for LexA, the type I signal peptidases, and
the class A
-lactamases(6, 7, 8, 9) .
Site-directed mutations were constructed in the tsp gene of pKK101 using the PCR mutagenesis technique of Higuchi(11) , and confirmed by DNA sequencing. Variants were purified in the same manner as wild type, except that it was sometimes necessary to elute more acidic variants of Tsp from the Q-Sepharose column using 10 mM Tris-HCl (pH 8.0), 300 mM KCl. Wild-type Tsp and the G375A, G376A, E433A, T452A, and K455A variants were also purified without denaturation to ensure that the properties of the purified proteins did not depend on the method of purification. In these cases, cells were grown, induced, and harvested as described above and resuspended in 10 volumes of a sphereoplasting buffer containing 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, 500 mM sucrose. After resuspension, the cells were collected by centrifugation and resuspended in 10 volumes of cold water to burst the outer membrane, and the cytoplasmic and membrane portions were removed by centrifugation. The soluble periplasmic fraction was brought to a final concentration of 10 mM Tris-HCl (pH 8.0), 100 mM sodium phosphate, 20 mM imidazole by dilution and addition of concentrated buffer, and chromatographed over a 2-ml nickel-nitrilotriacetic acid column. Tsp or its variants were eluted with loading buffer plus 250 mM imidazole, dialyzed against 10 mM Tris-HCl (pH 8.0), 100 mM KCl, and chromatographed over a Q-Sepharose column as described above. In each case where Tsp or its variants were purified under native conditions, their proteolytic activities in vitro and CD spectra were indistinguishable from those of the same proteins purified under denaturing conditions.
The
105 variant of the N-terminal domain of -repressor, a good protein
substrate for Tsp(1) , was purified by ion-exchange
chromatography, affinity chromatography, and gel filtration as
described previously(12) .
The CD spectra of Tsp or variants at concentrations of 50 µg/ml were recorded at 5 °C (to prevent autodegradation), in buffer containing 10 mM Tris-HCl (pH 8.0), 10 mM NaCl, using an Aviv model 60DS circular dichroism spectrometer.
Figure 2:
Substrate cleavage by wild-type Tsp and
selected variants. A, gel assays for degradation of the
-repressor 105 substrate by wild-type Tsp and variants after a
20-h incubation at 37 °C. B, effects of incubation of
wild-type Tsp or the S430C variant with 10 mM iodoacetamide or
10 mMN-ethylmaleimide for 20 min at room temperature
prior to the cleavage assay. Arrows indicate the positions of
the intact Tsp and
-repressor substrate bands. Approximately the
same initial concentration of Tsp or variant was used in each assay.
The final concentrations of enzyme are lower for wild-type Tsp and the
S430C and D441N variants because of autodigestion. Some Tsp variant
bands appear to have different mobilities because they were
electrophoresed on gels run for different periods of time. C,
time courses of degradation of Arc repressor (10 µM) by
wild-type Tsp and variants (250 nM) at 37 °C monitored by
loss of CD signal at 222 nm. D, effects of incubation of
wild-type Tsp or the S430C variant with 10 mM iodoacetamide
and 18.5 mM ethanol for 20 min at room temperature prior to
the Arc cleavage assay (conditions as in panel C). Control
reactions in which the protease is incubated with 18.5 mM ethanol alone are shown. Incubation with ethanol alone results in
a reduction in activity of about 5% for both wild-type Tsp and
S430C.
Figure 3: Circular dichroism spectra of wild-type Tsp and variants. A, spectra of wild-type Tsp and structurally unperturbed variants; B, spectra of wild-type Tsp and structurally perturbed variants.
As an assay for substrate binding, wild-type Tsp and the S430A, D441A, and K455A mutants were incubated with FITC-labeled insulin B chain, and the fluorescence spectra were recorded. As shown in Fig. 4, the spectrum of the FITC-labeled substrate shows decreased intensity and a small red shift in the presence of each of these proteins. No significant change in intensity or red shift is observed when FITC-labeled insulin B chain is incubated with the structurally perturbed G376A mutant or when the fluorescein dye is incubated with wild-type Tsp. These results suggest that the S430A, D441A, and K455A variants bind substrate in a fashion similar to wild-type Tsp, making it likely that the side chains altered by these mutations are involved directly in the catalytic mechanism.
Figure 4: Fluorescence emission spectra of FITC-labeled insulin B chain in the presence and absence of wild-type Tsp and variants. 667 nM of wild-type or variant Tsp was incubated with 667 nM FITC-labeled insulin B chain at 5 °C in buffer containing 10 mM Tris-HCl (pH 8.0) plus 20 mM NaCl, and the emission spectra were measured after excitation at 496 nm. Substrate binding is indicated by a reduction in intensity and a small red shift.
To determine if the carboxyl group of Asp-441 is required for activity, the isosteric D441N variant was constructed and purified. This mutant retains approximately 10% of the wild-type activity in vitro and is active in vivo (Fig. 2, Table 1). This result indicates that the carboxylate of Asp-441 is not essential for the proteolytic activity of Tsp. Since the D441A mutant is inactive, we presume that Asn-441 can partially substitute for Asp-441 by participating in similar hydrogen bonding or steric interactions.
To probe the tolerance of position 455 to substitutions, the codon was randomized, and 47 colonies were isolated, tested for activity in vivo, and sequenced. Although all residues except Met, Cys, and Trp were recovered, only genes encoding Lys at position 455 were active. The K455R and K455H variants were purified and found to be inactive when assayed for proteolytic activity in vitro (Fig. 2, Table 1). These data indicate that other basic side chains cannot functionally substitute for Lys-455. The K455H variant has a wild-type CD spectrum, while the K455R variant has an altered CD spectrum. The structural disruption caused by the seemingly conservative K455R substitution may indicate that the Lys-455 side chain is partially buried in a way that does not allow larger side chains to be accommodated.
In this study, we have used site-directed mutagenesis to identify residues important for Tsp activity in vitro and in vivo. Ser-430, Asp-441, and Lys-455 have properties expected of active site residues. Alanine substitution mutations at these positions abolish activity, but do not appear to affect substrate binding or to perturb the Tsp structure. Another group of residues (Gly-375, Gly-376, Glu-433, and Thr-452) seem to be important for maintaining the structure of Tsp. 13 additional residues, including each of the histidine residues in Tsp and two highly-conserved arginines, do not appear to be functionally or structurally important.
The three active site residues in Tsp appear similar in some respects to the residues of the catalytic triad of classical serine proteases. The traditional serine-protease mechanism involves a serine nucleophile, a histidine that acts as a general base to activate the hydroxyl group of the serine and an aspartate, which stabilizes the partial charge assumed by the histidine(19) . Substitution of any of the catalytic triad residues with alanine in well-studied serine proteases such as subtilisin reduces activity by more than 1000-fold (20) . The inactivity and partial activity, respectively, of the S430A and S430C mutants of Tsp, are consistent with the possibility that Ser-430 acts as a nucleophile. Moreover, although Tsp is not sensitive to inhibitors of classical serine proteases, such as DFP and phenylmethylsulfonyl fluoride, the S430C variant is inhibited by modification of the thiol group. Lys-455 of Tsp could function as a general base, in place of histidine, to activate the serine. However, Asp-441 of Tsp must play a role somewhat different than the aspartate of the classical catalytic triad, since the D441N substitution in Tsp does not affect activity, whereas the analogous substitution in trypsin reduces activity almost completely(21) .
Tsp may be similar,
in some respects, to the class A -lactamases, which use a serine,
lysine, and asparagine in hydrolysis of the lactam bond of penicillins
and cephallosporins(9) . Biochemical and crystallographic data
suggest that the serine of
-lactamase is activated for
nucleophillic attack by donating a hydrogen bond to the lysine, which
in turn is stabilized by hydrogen bonds with an asparagine and another
serine(9) . There is also evidence for mechanisms involving a
serine activated by a lysine for the bacterial proteases LexA, Lep
(EC), and SipS(6, 7, 8) . Like Tsp, these
proteases have critical serine and lysine residues, but they are not
readily reactive with phenylmethylsulfonyl fluoride or DFP. It is
important to note that although Tsp resembles these proteases and
-lactamases in some ways, it does not appear to share sequence or
structural homology with any of these enzymes.
The conservation of the critical serine, aspartate, and lysine residues of Tsp in CtpA, and the other sequences shown in Fig. 1suggests that the corresponding residues in these proteins will also be catalytically important. In the case of the IRBP repeats, two of the eight known sequences have a serine and aspartate at positions corresponding to Ser-430 and Asp-441 of Tsp. However, none of the IRBP repeats contains a lysine or other basic residue at positions corresponding to Lys-455 of Tsp. This lack of conservation of key residues is consistent with the fact that proteolytic activity has not been observed for IRBP. Instead, it appears that IRBP retains the fold of the Tsp active-site region, without the catalytic residues.