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J. Biol. Chem., Vol. 278, Issue 36, 34061-34065, September 5, 2003
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¶ || || ¶ ¶ ** ¶ 
From the
Department of Chemistry and Biochemistry,
Molecular Biology Institute, and the
¶UCLA-Department of Energy Institute for Genomics
and Proteomics, UCLA, Los Angeles, California 90095-1570
Received for publication, May 19, 2003 , and in revised form, June 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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9.4 and 7.0,
respectively. These measurements are inconsistent with the existence of a
thiolate-imidazolium ion pair and suggest a general base catalysis mechanism
during transpeptidation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The structure of SrtA revealed a novel protein fold and localized the active site to a hydrophobic surface depression that contains two highly conserved and enzymatically important residues, His120 and Cys184 (SrtA numbering) (16–18). This catalytic dyad is reminiscent of the active sites of the papain cysteine proteases (Cys25-His159-Asn175, papain numbering) (19, 20), suggesting that they are mechanistically related (21). In this model of SrtA function, the imidazole ring of His120 promotes the formation of the Cys184 thiolate, which then nucleophilically attacks the carbonyl carbon at the scissile Thr-Gly peptide bond in the LPXTG motif (22). After covalent linkage via a thioacyl bond to the threonine carbonyl group, the incoming amine of the cell wall precursor lipid II (23, 24) may then be deprotonated by His120 for attack on the covalent intermediate (16, 18).
Previous work left unresolved how His120 activates
Cys184. In the papain cysteine proteases, the cysteine side chain
is held in an active configuration through a thiolate-imidazolium ion
interaction with the histidine
(25). However, in the NMR
structure of SrtA solved in the absence of its substrates, the side chains of
Cys184 and His120 do not interact, arguing against the
presence of an ion pair (16).
To resolve this issue, and as a first step toward the design of a
therapeutically useful anti-infective agent, we have synthesized a
peptidyl-vinyl sulfone substrate mimic that inhibits SrtA. The pH dependence
of SrtA inhibition and NMR studies preclude the presence of an ion pair in the
active site, because His120 and Cys184 have
pKa values of 7.0, and 9.4, respectively.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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N59) and a single amino acid mutant of the
protein containing a cysteine to alanine substitution at position 184
(C184ASrtAN59) were overexpressed from
plasmids pSRTA and pHTT45, respectively
(16,
18). The expression, uniform
isotopic labeling (where applicable), and purification of
SrtAN59 and
C184ASrtAN59 have been described
previously (16). The
fluorescent substrate peptide d-QALPETGEE-e (where
d is dabcyl (4-([4-(dimethylamino)phenyl]azo)-benzoyl-) and
e is EDANS ([(2-aminoethyl)-amino]naphthlene-1-sulfonyl-)) was
purchased from Synpep (Dublin, CA) and purified by
HPLC.1 Reagents for
the synthesis of the vinyl sulfone inhibitor were purchased from Aldrich.
Synthesis of Vinyl Sulfone Inhibitor—The vinyl sulfone inhibitor was synthesized using solution phase methodology (see Fig. 1). The Leu-Pro-Ala tripeptide was synthesized by standard amino acid coupling chemistry using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI) and 4-(dimethylamino)pyridine (DMAP), starting with the carbobenzyloxy-protected amino acid Cbz-Leu-OH and N-tert-butoxycarbonyl (N-t-Boc) methyl esters of alanine and proline. L-Threonine was fully protected as the N-t-Boc-threonine methyl ester with the alcohol protected as the t-butyldiphenylsilyl ether (2) in three steps. The ester was reduced with diisobutylaluminum hydride (DIBAL-H) to the aldehyde, which was immediately reacted without purification with diethyl phenylsulfonylmethylphosphonate (3) to give the desired vinyl sulfone functionality (4) in good yield. Removal of the Boc group with trifluoroacetic acid followed by coupling the amine with the Cbz-protected tripeptide gave the desired tetrapeptide. Removal of the t-butyldiphenylsilyl group with HF gave the vinyl sulfone inhibitor (1). The inhibitor was purified by silica gel chromatography and the structure confirmed by 1H and 13C NMR spectroscopy and FAB+ (fast atom bombardment) mass spectrometry.
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Enzyme Assays—Fluorescent measurements of SrtA activity in
the presence of the vinyl sulfone inhibitor were performed in 96-well
microtiter plates as described previously
(26). Inhibition reactions
contained 5 µM SrtA and vinyl sulfone inhibitor (100, 300, or
600 µM) in buffer R (50 mM Tris, pH 3.5–10, 150
mM NaCl, 5 mM glycine, 5 mM
CaCl2). d-QALPETGEE-e was dissolved in dimethyl
sulfoxide and added to the reaction at a final concentration of 25
µM, for a total reaction volume of 200 µl. The final
concentration of Me2SO in the reaction was kept below 5%. The
reactions were incubated for 12 h at 37 °C with gentle mixing in a GENios
multiwell fluorimeter (Tecan, Durham, NC) (360 nm excitation filter, 465 nm
emission filter). Fluorescence emission was recorded at 10-min intervals and
blanked against a reference solution containing SrtA in buffer R. All enzyme
assays were performed in triplicate, resulting in 450 measurements for
each inhibitor concentration assayed. The inhibition parameters
Ki and ki were solved simultaneously
for each inhibitor concentration using the equation,
 | (Eq. 1) |
HPLC Analysis of Inhibitor Modification of SrtA—Thirty microliters of SrtA (95–100 µM) in buffer I (50 mM Tris-HCl, 150 mM NaCl, and 5 mM CaCl2) were adjusted to the desired pH with 1 M HCl or 1 M NaOH as needed and were incubated with a 20-fold molar excess of the vinyl sulfone inhibitor for 20 h at 37 °C with gentle agitation. The reaction was stopped by adding 500 µl of buffer A (0.1% trifluoroacetic acid in water), and the products were separated by reverse phase HPLC using a C18 column (Waters, Milford, MA) with the application of a gradient of 25–50% buffer B (90% acetonitrile, 10% H2O, 0.1% trifluoroacetic acid).
NMR Spectroscopy—NMR experiments were carried out at 308 K
on a Bruker DRX500 spectrometer equipped with a triple resonance probe using
15N- and 13C-labeled
SrtAN59 and
C184ASrtAN59 proteins (0.5
mM) in buffer N (50 mM Tris-HCl (pH 6.2), 100
mM NaCl, 20 mM CaCl2, 3 mM
dithiothreitol, and 7% D2O). The chemical shifts of wild-type
SrtAN59 have been reported previously, and the
resonances of the single histidine side chain (His120) in the
C184ASrtAN59 mutant were readily
assigned by reference to these data. For the pKa
measurements, a series of two-dimensional 1H-13C
heteronuclear single quantum correlation spectra were recorded to monitor the
pH dependence of the 1H-
2-13C-2 and
1H-
1-13C-1 resonances of the
His120 side chain (spectra recorded at pH values of 4.5–10).
Chemical shifts in the NMR spectra were plotted as a function of pH and fit to
the following equation,
 | (Eq. 2) |
HA and A are the chemical shifts of
the fully protonated and deprotonated forms of the ionizable group, and
obs is the observed chemical shift
(28,
29). The data were
fit using SigmaPlot2000. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The efficacy of the vinyl sulfone compound was tested in vitro by
determining how it altered the SrtA-catalyzed hydrolysis of an internally
quenched fluorescent substrate analogue
(d-Gln-Ala-Leu-Pro-Glu-Thr-Gly-Glu-Glu-e). Hydrolysis
progress curves were generated by monitoring the increase in fluorescence that
accompanies the cleavage of the substrate
(18,
26). The curves were
consistent with the vinyl sulfone compound acting as an irreversible inhibitor
of SrtA (Fig. 2A)
(35). Typical progress curves
of the reaction in the presence of 100, 300, and 600 µM
inhibitor are shown in Fig.
2B. Fits of these data to
Equation 1 by non-linear
regression analysis determined the first-order rate constant of inactivation
(ki) and the dissociation constant of inhibitor binding
(Ki). At pH 7, the Ki of the inhibitor
is 9 x 10–6 M, which is
comparable with the measured Km of SrtA of 3 x
10–6 M for the fluorogenic substrate
analogue (data not shown). The rate constant of inactivation
(ki) of the vinyl sulfone inhibitor is 4 x
10–4 min–1. To date,
only two other irreversible SrtA inhibitors have been characterized. Both
contain the Cbz-Leu-Pro-Ala-Thr sorting signal mimic of the vinyl sulfone
compound but utilize different reactive groups (diazomethane and
chloromethane) (26). The
first-order rate constants of the peptidyl-diazomethane (5.8 x
10–3 min–1) and
-chloromethane (1.1 x 10–2
min–1) SrtA inhibitors are larger than the vinyl
sulfone ki by 10- and 20-fold, respectively
(26). This difference in
reactivity is consistent with the higher electrophilicity of chloro- and
diazomethane reactive groups and has been observed in inhibition studies of
other cysteine proteases (36).
For example, diazomethane and chloromethane inhibitors of human cathepsin L
have second-order rate constants up to 100-fold higher than vinyl sulfone
inhibitors (30,
36). As the transpeptidation
reaction of SrtA is extremely poor in vitro
(kcat/Km) = 20.6
M·s–1 it probably does not
reflect the efficiency of the enzyme in vivo, where it must complete
the anchoring of surface proteins within the doubling time of the bacterium.
The development of a quantitative in vivo assay for inhibition will
be necessary to evaluate the efficacy of peptidyl inhibitors of sortase as
anti-infective agents.
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Because the vinyl sulfone warhead of the peptide inhibitor is expected to
be most reactive toward cysteine thiolates, and Cys184 is the only
cysteine in SrtA, we measured the pH dependence of inactivation to determine
the pKa of Cys184. The in
vitro hydrolysis reaction was repeated at the three inhibitor
concentrations over the pH range of 3.5–10, and the inhibition
parameters Ki and ki were determined
by curve fitting. As shown in Fig.
2C, ki increases dramatically above
pH 8, whereas the Ki value is not significantly affected.
To estimate the pKa of Cys184, a
modified Henderson-Hasselbach equation
(37),
 | (Eq. 3) |
9.4.
To confirm that the pH-dependent inhibition of SrtA results from the
covalent modification of Cys184, we tracked the modification
reaction by chromatography. The SrtA protein was incubated with a 20-fold
molar excess of inhibitor for 20 h, and the reaction products were separated
by HPLC. Fig. 3,
A–C shows the results of incubating SrtA
with the inhibitor at pH 9, 7, and 6, respectively. Two inhibitor-modified
SrtA species elute on a C18 reverse phase HPLC column (peaks 1 and
2), and mass spectrometry indicates that both peaks contain SrtA
covalently modified by a single inhibitor molecule. At pH values of 6 and 7,
little modification occurs within 20 h with a slight excess of species 2 being
produced. However, at pH 9.0, nearly all of the SrtA protein is converted to
species 1 (Fig. 3A).
An analysis of the pH dependence of modification indicates that the production
of species 1 is most efficient at pH values nearing 9, whereas maximal
production of the minor species 2 occurs at pH 7 (data not shown).
Because the enzyme is maximally inhibited by the vinyl sulfone compound at
high pH values (Fig.
2C), where species 1 is almost exclusively present, the
data strongly suggest that species 1 corresponds to the inhibitor-SrtA complex
that forms during the inactivation. This hypothesis was substantiated by
thiolating Cys184 with a 2-fold excess of nitroxide spin label
HO-225 ((1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate)
(38) and testing for its
ability to react with the vinyl sulfone inhibitor at pH 9. As shown in
Fig. 3D, preincubation
with HO-225 prevented the formation of both species 1 and 2. These data are
consistent with Cys184 being the primary and most reactive site for
modification by the vinyl sulfone at pH values above 8.
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To investigate whether an imidazolium ion is present in the active site of
SrtA, NMR was used to determine the pKa of the
His120 side chain. Because the chemical shifts of atoms within the
imidazole are expected to be sensitive to the ionization state of the side
chain, a series of 1H-13C heteronuclear single quantum
correlation spectra were recorded using a 13C- and
15N-enriched sample of SrtA, and the chemical shifts of the
1H-2 and 1H-1 atoms of His120
were recorded at pH values between 4.5 and 10.
Fig. 4 shows a plot of the
chemical shift of the 1H-1 atom as a function of pH (a
similar curve was obtained for the 1H-2 atom). A fit of the
titration data to Equation 2 (see
"Experimental Procedures") indicates that the His120
side chain has a pKa of 7.0 ± 0.1 in the presence
of 20 mM CaCl2. Because Ca2+
binding near the active site has been shown to enhance the in vitro
activity of SrtA 8-fold (16),
we repeated the pH titration experiment in calcium-free buffer (buffer N with
no calcium) to determine the effects of Ca2+ on the
ionization state of the active site His120 residue. No significant
changes in the pKa of His120 were detected in
the absence of calcium (pKa = 6.6 ± 0.1)
(Fig. 4B). The data
indicate that at physiological pH, the imidazole side chain is only partially
protonated and that calcium does not stimulate the activity of SrtA by
altering the ionization state of His120. Ca2+
binding may stabilize substrate binding or the fold of the protein.
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If a thiolate-imidazolium ion pair exists in the active site of SrtA, one
would expect the thiolate of Cys184 to perturb the ionization of
His120 (i.e. alter its pKa). We
ascertained the effect of Cys184 on His120 by repeating
the NMR-pH titration experiment using the mutant
C184ASrtAN59. The results of this
titration (Fig. 4A,
open circles) were fit to Equation
2 to obtain a pKa of 7.3 ± 0.1. The
similarly measured pKa values for His120 in the
wild-type and mutant SrtA proteins suggest that its ionization state is
independent of the Cys184 side chain, a finding that is
inconsistent with the existence of an ion pair between His120 and
Cys184.
A thiolate-imidazolium ion pair between the side chains of His120 and Cys184 would enhance the reactivity of the thiol toward electrophiles at weakly acidic and neutral pH (36) because the imidazole group would act to polarize the thiol. In this scenario, it is expected that the pKa value of the cysteine thiol would be more acidic and the histidine imidazole would be more basic than their normal values of 7.85 and 6.0, respectively (39). For example, the anomalous pKa values of 3.3 (for the Cys25 thiol) and 8.5 (for the His159 imidazolium) support the existence of a thiolate-imidazolium ion pair in papain (40, 41). In SrtA, however, our investigation argues against the use of a thiolate-imidazolium ion pair in the reaction mechanism.
The pH dependence of modification of Cys184 by the vinyl sulfone
inhibitor (Fig. 2C)
shows a dramatic increase in the first-order rate of inactivation as the pH is
raised from 7.5 to 10 (at pH 10 the inhibitor is 20 times more reactive than
at pH 7.5). Because the inhibitor is expected to be more reactive toward a
thiolate ion, the pH dependence of inhibition can be attributed to the
deprotonation of Cys184. These data argue against the presence of
an ion pair in the active site, because the pKa
of Cys184 is estimated to be 9.4.
NMR studies of the His120 side chain have measured its
pKa at 7.0 in the wild-type protein, which is inconsistent
with the presence of an imidazolium cation at neutral pH. Moreover, our
finding that the removal of the Cys184 side chain
(C184ASrtAN59) has only a modest
effect on the ionization state of His120 side chain argues against
the presence of an ion pair, because in the papain system the
pKa of His159 is lowered by 4.5 pH
units upon the methylthiolation of the Cys25
(25).
Although the thiolate-imidazolium ion pair is a common catalytic entity of cysteine proteases, it is not universal (42, 43). The absence of an ion pair in SrtA suggests its catalytic mechanism may be similar to the viral 3C proteases (picornains), a structurally and mechanistically distinct group of cysteine proteases that perform general base catalysis (reviewed in Refs. 36 and 44). The crystal structures of the picornains of hepatitis A (45), rhinovirus (46), and poliovirus (47) show a similarity of three-dimensional structures and catalytic mechanisms to the serine proteases of the trypsin/chymotrypsin family and may be evolutionarily related (36, 43, 44). The pH-dependent alkylation of the active site cysteine of poliovirus protease 3C with iodoacetamide has measured its pKa at 8.86 (42), which is similar to the estimated pKa of the SrtA thiol. This is consistent with a reaction mechanism in which the cysteine nucleophile is uncharged at physiological pH, and the histidine functions as a general base.
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FOOTNOTES |
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|| Both authors contributed equally to this work.
** To whom correspondence may be addressed. Tel.: 310-825-7954; Fax: 310-206-3722; E-mail: jung{at}chem.ucla.edu.
To whom correspondence may be addressed. Tel.: 310-206-2334; Fax:
310-206-4749; E-mail:
rclubb{at}mbi.ucla.edu.
1 The abbreviations used are: HPLC, high pressure liquid chromatography;
N-t-Boc, N-tert-butoxycarbonyl; Cbz,
benzyloxycarbonyl.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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