From the Istituto di Ricerche di Biologia Molecolare "P.
Angeletti," Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy
The NS3 region of the hepatitis C virus encodes for a
serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with
proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines
(Cys97, Cys99, and Cys145)
and one histidine residue (His149), which is postulated to
coordinate the metal through a water molecule. In this article, we
present an analysis of the role of metal coordination with respect to
enzyme activity and folding. Using NMR spectroscopy, the resonances of
His149 were assigned based on their isotropic shift in a
Co(II)-substituted protein. Data obtained with 15N-labeled
NS3 protease were compatible with the involvement of the
-N of
His149 in metal coordination. pH titration experiments
showed that the cooperative association of at least two protons is
required in the protonation process of His149. Changes in
the NMR signals of this residue between pH 7 and 5 are interpreted as
evidence for a structural change at the metal binding site, which
switches from a "closed" to an "open" conformation. Site-directed mutagenesis of His149 has shown the
importance of this residue in the metal incorporation pathway and for
achieving an active fold. The metal coordination of the protease was
also investigated by circular dichroism and electronic absorption
spectroscopies using a Co(II)-substituted enzyme. We show evidence for
rearrangements of the metal coordination geometry induced by complex
formation with an NS4A peptide cofactor. No such changes were observed
upon binding to a substrate peptide. Also, CN
and
N3
induced Co(II) ligand field perturbations, which
went along with an 1.5-fold enhancement of protease activity.
 |
INTRODUCTION |
The hepatitis C Virus (HCV)1
has been identified as the major etiologic agent of parenterally
transmitted non-A non-B hepatitis (1, 2). HCV is an enveloped virus
with a positive-stranded RNA genome of 9.4 kb, which is translated into
a precursor polyprotein of about 3010 amino acids (3). Both cellular
and virally encoded proteases are involved in the maturational
proteolytic processing of this precursor. Whereas the structural viral
proteins arise from signal peptidase-catalyzed cleavages (4), two
different proteolytic activities encoded by the HCV NS2 and NS3
proteins are responsible for the processing of the nonstructural region of the polyprotein. The NS2-NS3 precursor is cleaved intramolecularly by an autoprotease, the activity of which was shown to be
zinc-dependent (5). The N-terminal part of the NS3 protein,
furthermore, contains a 20-kDa serine protease domain that accomplishes
all cleavage events downstream of NS3, including the generation of the
mature viral polymerase (6). In order to perform its physiological task, the NS3 serine protease has to bind to the viral protein NS4A (7,
8). This binding event leads to an enhancement of the protease activity
and to a stabilization of NS3. In vitro, activation of NS3
can be achieved by addition of peptides harboring residues 21-34 of
NS4A (9-12).
Based on a homology model, we were able to predict the presence, in the
NS3 protease domain, of a tetradentate metal binding site formed by
three cysteines (Cys97, Cys99, and
Cys145) and one histidine residue (His149)
(13). Biochemical characterization has confirmed this prediction and
demonstrated the presence of a zinc ion in a tetrahedral environment (13, 14). This zinc ion was shown to be essential for the structural
integrity of the protein; its removal leads to unfolding and
aggregation of the enzyme. Mutagenesis experiments have shown that
mutations affecting any of the three cysteine residues resulted in an
impaired NS3 protease activity as judged from in vitro
translation experiments (14, 15). On the other hand, mutagenesis of
His149 into alanine had only minor effects on the
autoprocessing of NS3-containing precursor polyproteins (14, 15). It is
presently not clear whether the zinc ion of the NS3 protease is
identical to the zinc that has been shown to be essential for the
NS2-NS3 autoprotease activity.
Crystal structures of the NS3 protease domain (16) and of the complex
with an NS4A cofactor peptide (17) have been published. Both structures
confirm the prediction of the metal binding site and precisely locate
it on the surface of the protein, well exposed to the solvent. In the
structure of the NS3-cofactor complex the zinc ion is coordinated to
the three predicted cysteine ligands and, through a water molecule, to
His149. The indirect interaction between the metal and the
histidine ligand is consistent with the weak effects of mutations in
this position. On the other hand, coordination by His149
through a water molecule is observed only in two of the three monomers
in the asymmetric unit of the crystals obtained in the absence of the
NS4A peptide (16). In the third monomer, the His149-N
moves away from the zinc and thus does not participate in the
coordination. The precise function of water and the influence of the
cofactor on the coordination sphere of the zinc atom are still open
issues because the metal binding site is located on the protein
surface, and crystal packing is likely to play a role. Hence, the
interest in further investigations of the solution state.
In the present study, we have investigated zinc- and cobalt-substituted
NS3 protease using NMR, CD, and visible spectroscopy. We present
evidence for pH-dependent changes in the metal coordination by His149 that are compatible with the presence of a
bridging hydroxyl group as metal-ligand. We furthermore show that the
coordination geometry of the metal may undergo different conformational
rearrangements that influence enzymatic activity.
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MATERIALS AND METHODS |
Purification of NS3 Protease Domain--
A plasmid containing
the serine protease domain of NS3 (amino acids 1-180, from HCV Bk
strain, followed by the sequence ASKKKK) cDNA under the control of
the bacteriophage T7 gene 10 promoter was used to transform
Escherichia coli BL21(DE3) cells (18). Protein expression
and purification was carried out as described previously (13).
15N-Labeled NS3 was prepared using
(15NH4)2SO4 in a
minimal medium containing 100 mM potassium phosphate, pH
7.0, 0.5 mM MgSO4, 0.5 mM
CaCl2, 13 µM FeSO4, 50 µM ZnCl2, 7 µM thiamine, 6 µM biotin, and glucose (4 g/liter). Purity of the enzyme
was evaluated to be >95% by silver-stained SDS-polyacrylamide gels
and by reversed-phase high performance liquid chromatography using a
Vydac C4 column (4.6 × 250 mm, 5 µm, 300 Å). In the latter case, eluents were H2O, 0.1% trifluoracetic acid (eluent
A) and acetonitrile, 0.1% trifluoroacetic acid (eluent B). A linear
gradient from 3 to 95% eluent B in 60 min was used. The concentration
of protein stocks was estimated by quantitative amino acid
analysis.
Synthesis of Co(II) NS3 Proteases--
Co(II)-containing
recombinant wild type and S139A NS3 proteases were biosynthetically
prepared as described previously (13). Contents of cobalt and
adventitious zinc were determined on nitric acid-hydrolyzed proteins by
atomic absorption spectroscopy on a Perkin-Elmer model 2100 atomic
absorption spectrometer equipped with a graphite furnace. Standardized
Zn2+ and Co2+ solutions were purchased from
Merck. Glassware used for metal analysis was washed with 50% nitric
acid and thoroughly rinsed with Chelex-100-treated deionized water.
Wild type and S139A Co(II) proteins had a protein:metal stoichiometry
of 1:0.91 ± 0.02 and 1:0.95 ± 0.03, respectively.
NMR Spectroscopy--
NMR spectra of Zn NS3 were measured with
Bruker AMX 400 (one-dimensional experiments) and AMX 500 (two-dimensional experiments) instruments. Samples (0.4-0.6
mM) were brought into the 2H2O
solvent system by extensive dialysis against 99%
2H2O buffer 4%
2H5-glycerol, 0.1% CHAPS, 3 mM
DTT, 10 mM sodium phosphate, 10 mM MES, pH 6.5, at 4 °C under N2. Protein samples were let exchange protons for 5 days before any measurements were acquired. The presence
of glycerol in solution greatly enhances the viscosity, so that
20 °C was established to be the lower limit. On the other hand, the
enzyme activity is greatly enhanced above 30 °C, so the working
temperature of 25 °C was set by a compromise between these two
restricting factors.
For the pH titration experiments, protein samples were dialyzed for
2 h against 20 volumes of the same buffer with a slightly different pH. The pH was measured by a PHM84 Radiometer-pH meter equipped with an Aldrich calomel microelectrode. The one-dimensional titration experiments were acquired with a SW of 4800 Hz and 8K complex
data points. No water presaturation was necessary. The data were
analyzed with the SWAN-MR software (19). The pKa deviation due to the deuterium isotope effect was estimated to be +0.05
pH units, comparing the pH and p2H profile of the imidazole
1H in the same protein buffer system. Data reported below are
already corrected for this difference. Data were fitted using the
following equation,
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(Eq. 1)
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or, with allowance for cooperativity,
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(Eq. 2)
|
where ppm is the observed chemical shift, a and
b are the asymptotic values, and n is the Hill
coefficient.
A line width analysis of peak C, identified as His149, in
the pH range 5.0-8.0 was carried out assuming the following minimal model,
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(Eq. 3)
|
where C and C+ represent the imidazole moiety in the
unprotonated and protonated forms, respectively, and
k+1 and k
1 are the
kinetic constants of the process. The rate constant for the
deprotonation reaction, k
1, was estimated from
the dependence of the apparent spin-spin relaxation times on the molar fraction of the two His species described by the following equation (20),
|
(Eq. 4)
|
where (1/T2C)obs and
(1/T2C+)obs are the observed line
widths at different pH values, 1/T2C and
1/T2C+ are the spin-spin relaxation times in
absence of exchange, and (fC+/fC) is
the ratio of the respective molar fractions.
Two TOCSY experiments (
m = 28 and 48 ms), at several different
pH values, were acquired, with a SW of 6000 Hz (t2) × 6000 Hz (t1) and
1K × 256 complex data points. No water presaturation was
necessary. The time dependence of the exchange process was further
investigated at pH 6.0 with two-dimensional NOESY spectroscopy. A set
of experiments was acquired, with mixing times of 10, 30, 60, 100, and
200 ms. The SW values were 6000 Hz (t2) × 6000 Hz (t1) and 1K × 256 complex data points. All the experimental matrices were identically
transformed with a Gauss to Lorentzian apodization function (t2) and a
70 degree shifted sine bell function (t1); the final dimensions after
transform were 2K (t2) × 1K (t1). All the two-dimensional experiments
were analyzed using the nmrPipe (21) and NMRView (22) programs on an
Indigo2 SGI workstation. The kinetic process was analyzed following the
procedure outlined by Clore et al. (23) that will be briefly
summarized here. The time development of magnetization in the system
described by Equation 3 is given by the following equation.
|
(Eq. 5)
|
The equilibrium constant of the process, at pH 6.0, is defined
as K = k+1/k
1 = [C+]/[C], where the concentration of hydrogen ion has
been incorporated in the constant; RC and
RC+ are the total spin lattice relaxation rates
of the relevant protons. At time 0, the intensity of the diagonal peak
of species C is set to 1 and the cross-peak to 0, whereas the
equilibrium magnetization (MCe
and MC+e) is expressed as a
function of the constant rates by a trivial rearrangement of the mass
conservation law. The build-up curves were fitted simultaneously to the
differential equations (Equation 5), under the control of a nonlinear
least squares optimization routine varying the values of the
dissipative rates and of the kinetic constants, using the program MLAB
(Civilized Software, Bethesda, MD).
The Co(II)-substituted NS3 samples in 2H2O, for
the NMR measurements, were prepared by H
2H exchange of
protein samples loading the purified protein (10 mg) on an Amersham
Pharmacia Biotech HR 5/5 Mono S column equilibrated with
H2O buffer (4% glycerol, 0.1% CHAPS, 10 mM
sodium phosphate, pH 7.5). The protein was then washed with the same
buffer in 99% 2H2O (buffers were flushed with
dry nitrogen) and eluted by a steep NaCl gradient. The protein was
further concentrated to 0.4 mM and dialyzed twice against
20 volumes of 2H2O, 4%
2H5-glycerol, 0.1% CHAPS, 10 mM
sodium phosphate, pH 7.5, at 4 °C under argon. After storage for 2 days at 4 °C in 2H2O buffer, the protein
samples (0.3-0.4 mM) were analyzed by one-dimensional NMR
spectroscopy.
15N-filtered NOESY (60 and 100 ms) and ROESY (20 ms) were
acquired in water using the 15N-labeled sample, at pH 6.0 and 6.6. The 15N labeling was used to filter out of the
spectra all of the resonances originating from the amide groups of the
protein, so that in the region 7.5-9.0 ppm, only the resonances
originating from the
1H protons of the histidines were visible. The
SW and data treatment were the same as described previously. The
experiments were acquired using water flip back pulses combined with
z-gradients to avoid saturation of the water signal and to allow the
use of a convenient receiver gain (512 in our experiments).
1H-15N HSQC experiments were acquired at pH 6.3 and 6.6, using as transfer delays
m = (1/(4J) = 11.0 ms, to
obtain the coherence transfer from the
1H and
2H protons to the
2N and
1N through the 2J coupling constant. The SW
values were 6000 Hz and carrier at 7.8 ppm (t2) and 4700 Hz and carrier
at 200 ppm (t1). The experimental matrix was 1K × 80 complex data
points; it was transformed using a Gauss to Lorentzian apodization
function (t2) and a 62°-shifted sine bell function to yield a final
matrix of 2K × 512 data points.
H149A Mutant and Apo NS3 Protease--
The H149A mutation was
inserted by polymerase chain reaction site-directed mutagenesis using
suitable primers. Full sequencing of the mutated cDNA confirmed the
presence of a single mutation. Transformed E. coli BL21(DE3)
cells were grown at 37 °C and induced at an
A600 nm of 0.7-0.9 with 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C in LB medium. Under these conditions, the protease was found
in the insoluble fraction. The harvested cells were resuspended in 100 mM NaCl, 50 mM Tris-HCl, pH 8.0 (10% v/v), and
1 mg/ml lysozyme. After 20 min of incubation at room temperature, pellets were disrupted in a French pressure cell. The insoluble material was collected by centrifuging the homogenate at 5000 × g for 10 min at 4 °C. It was then resuspended in ice-cold
100 mM NaCl, 1 mM EDTA, 0.1% sodium
deoxycolate, and 50 mM Tris-HCl, pH 8.0. After 10 min of
mixing, 8 mM MgCl2 was added, together with 10 µg/ml DNase I. Digestion was carried out for 45 min, followed by 10 min of centrifugation at 10,000 × g. Pellets were
resuspended and centrifuged in 1% Nonidet P-40, 100 mM
NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, and
washed in 1 M urea, 50 mM Tris (pH 8.0). This
procedure was repeated twice before dissolving the pellets in 8 M urea and 60 mM DTT overnight at 4 °C. The
dissolved material contained protease with a purity >80%. Further
purification to >90% purity was done on a Amersham Pharmacia Biotech
Superdex 75 HiLoad 26/60 gel filtration column, equilibrated in 6 M urea, 30 mM DTT and 100 mM sodium
phosphate, pH 7.5. The gel-filtered protein (1 µM) was
dialyzed against 1% trifluoroacetic acid, 500 mM urea for
16 h and against 0.1% trifluoroacetic acid for a further 18 h before being concentrated to 25 µM, nitrogen shock
frozen, and stored at
80 °C. Protein stocks were quantified by
amino acid analysis, and the purity grade was assessed as described previously.
Apo wild type NS3 was obtained by denaturing purified zinc protein in 8 M urea, 30 mM DTT, and 10 mM EDTA.
Two steps of dialysis against trifluoroacetic acid as described
previously were applied to remove urea. Then zinc content of the acid
denatured enzymes has been estimated to be less then 1% of the total
protein concentration. Proteins were refolded before being assayed in
50 mM Tris, pH 7.5, 2% CHAPS, 50% glycerol, 10 mM DTT (standard activity buffer) and treated with
Chelex-100 resin before the addition of 10 µM ZnCl2.
Apo wild type (21 µM) and His149 proteins (25 µM) were diluted to 0.1-1 µM in a cuvette
containing standard activity buffer with different amounts of
ZnCl2 (1.5-100 µM) or 100 µM
EDTA. Upon dilution, fluorescence traces (
EX = 280 nm,
2.5 nm slits;
EM = 340 nm, 8 nm slits) were recorded
under continuos stirring on a Perkin-Elmer LS 50B fluorescence
spectrometer equipped with a cuvette holder, thermostatted at
22 °C.
Experiments were repeated in triplicate and analyzed with the help of a
Grafit software (Erithacus).
High Performance Liquid Chromatography Activity Assay and Active
Site Titration--
Peptide synthesis was performed by Fmoc
(N-(9-fluorenyl)methyloxycarbonyl)/t-Bu chemistry as
described previously on a NovaSyn Gem flow synthesizer (18, 24).
Concentrations of stock solutions of peptides, which were prepared in
Me2SO or in buffered aqueous solutions and kept at
80 °C until use, were determined by quantitative amino acid
analysis performed on HCl-hydrolyzed samples.
Standard cleavage assays, unless otherwise stated, were performed in 57 µl of 50 mM Tris, pH 7.5, 2% CHAPS, 50% glycerol, 10 mM DTT to which 3 µl of a synthetic peptide, derived from
the cleavage sequence of the NS4A-NS4B junction (S1 = DEMEEC-ASHLPYK), were added. DTT was omitted from buffers when assaying
Co(II)-NS3 and wild type enzyme for KCN perturbation. As a protease
cofactor, we used a 17-mer peptide corresponding to the central
hydrophobic core of the NS4A protein spanning residues 21-34 with an
N-terminal lysine tag (4A peptide, KKKGSVVIVGRIILSGR). Enzyme
concentrations (10 nM to 1 µM) and incubation
times were chosen in order to obtain <10% substrate conversion.
Reactions were quenched by addition of 40 µl 1% trifluoroacetic acid
and analyzed by high performance liquid chromatography on a Lichrospher
C18 reversed-phase cartridge column (4 × 125 mm, 5 µm, Merck)
using a 10-50% acetonitrile gradient at 5%/min. Peak detection was
accomplished by monitoring both the absorbance at 220 nm and tyrosine
fluorescence (
ex = 260 nm,
em = 305 nm).
Active site titrations were performed on an SX-MV18 Applied
Photophysics stopped flow apparatus in standard activity buffer. Under
these conditions, we measured a dead time of 10 ms with a standard
reference reaction (reduction of 2,6-dichlorophenolindophenol by
L-acorbic acid) (25). We used a fluorescence resonance
energy transfer based ester substrate (26, 24) having the sequence S2 = Ac-DED(EDANS)EE U
[COO]ASK(DABCYL)-NH2 to measure the initial
burst phase of ester hydrolysis. This is directly proportional to the
active site concentration (27). Fluorescence standard curves were
calculated either by measuring the fluorescence of known concentrations
of 100% hydrolyzed substrate or by reference to fully active wild type
protein (24). Wild type and H149A mutant proteases at concentrations of
50, 100, and 200 nM were incubated with of 80 µM 4A peptide and rapidly mixed with an equal volume of
50 µM S2. Fluorescence traces (ex = 355 nm) were recorded for 5 s with a logarithmic time scale
acquisition, using a 400-nm-cutoff filter. The averages of eight single
determinations were analyzed by extrapolating the linear phase to zero
time.
Electronic Absorption and Circular Dichroism
Spectroscopy--
80 µM of wild type Co(II) NS3 or 100 µM of the S139A mutant were incubated with different
amounts of 4A peptide or S1 in 50% glycerol, 2% CHAPS, 30 mM NaCl, and 20 mM sodium phosphate, pH 7.5. Sample concentrations were determined by quantitative amino acid
analysis. KCN and NaN3 additions were carried out using 40 µM Co(II) NS3 in presence of 150 µM 4A
peptide. Electronic spectra of proteases were acquired on a Varian Cary
3E dual-beam spectrophotometer. Spectra were recorded at 15 °C at 60 nm/min scan speed with 1-cm path length quartz cuvettes. Circular
dichroism measurements were performed using a Jasco 710 spectropolarimeter equipped with a cell holder thermostatically
controlled by a circulating water bath. Spectra were collected with a
8-s time constant and a 5 nm/min scan speed at 15 °C by using
rectangular quartz cells of 1 cm path length and a protein
concentration of 40 µM. The mean residue ellipticity Ø was calculated referring to the protein residues concentration. Data
were analyzed with the help of Kaleidagraph software (Abelbeck).
 |
RESULTS |
NMR Spectroscopy--
The frequencies arising from the
1H and
2H nonexchangeable protons of the three histidine residues were
identified in the two-dimensional TOCSY experiment through their
characteristic cross-peak correlation. In Fig.
1 the aromatic region of the TOCSY experiment
at pH 6.4 is shown. The His
1H
2H cross-peaks are labeled
A (
1H = 8.22;
2H = 7.10), B
(
1H = 7.87;
2H = 7.05), and C (
1H = 7.84;
2H = 6.88). The same lettering is held also in the
one-dimensional set of the experiments shown in Fig. 3a, where the pH behavior of the His signals is explored.

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Fig. 1.
Two-dimensional NMR TOCSY spectra of Zn NS3
protease aromatic region. Spectra were recorded in
2H2O buffer containing 4%
2H5-glycerol, 0.1% CHAPS, 1 mM
DTT, 10 mM sodium phosphate, pH 6.4. Histidine
1H- 2H cross-peaks are labeled with
letters; A corresponds to His57, B to
His110, and C to His149.
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|
To assign the His149
1H signal, we have used a
Co(II)-substituted protein. Co(II) has been used as a substitute for
zinc in many metalloproteins due to its similar coordination behavior, leading to very little alteration of the coordination geometry (28).
The paramagnetic nature of Co(II) isotropically shifts resonances of
the surrounding atoms (29-32). This effect permits the direct
identification of the protons in proximity to the paramagnetic metal
ion. The imidazole
1H signal of histidine residues involved in metal
coordination is generally shifted to the downfield region of the
spectrum (29).
One-dimensional spectra of Co(II) NS3 were obtained at pH 6.85 (Fig.
2). Experiments were also repeated at
different pH values using both the Zn- and the Co(II)-substituted
enzymes (Fig. 3a). In Fig. 2, it
can be seen that the signal at 7.84 ppm (peak C) was not present in the
Co(II) NS3 spectra and that a new broad peak was observed at 38.5 ppm
(peak C*). This shift in the downfield region is consistent with the
previously reported shifts of the histidine
1 proton of
Co(II)-substituted enzymes (30, 32) and with the imidazole spectra of
model complexes, such as Co(1Me-Imi)2Cl2 and Co(Imi)2Cl2 (29, 31). The
broad line width is characteristic of fast relaxing protons influenced
by distorted tetrahedral coordinations of Co(II) complexes (29, 32).
Enzyme preparations with different Co/Zn ratios were obtained by
controlled dialysis against zinc-containing buffer. The Co/Zn ratio
determined by atomic absorption spectroscopy correlated with the ratio
of the intensities between the peaks at 38.5 and 7.84 ppm (data not
shown). These findings assign the resonance of the
H of the
zinc-bound His149 to that of peak C.

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Fig. 2.
NMR spectrum of Co(II) NS3 protease. The
spectrum was recorded in 2H2O buffer containing
4% 2H5-glycerol, 0.1% CHAPS, 10 mM sodium phosphate, pH 6.85. The exponential line
broadening of the aromatic region was set to 1 Hz, and the downfield
region of Co(II) enzyme was processed with a 30 Hz exponential line
broadening function. Baselines were corrected by a fourth-order
polynomial function. The arrow indicates the position were
peak C is missing.
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Fig. 3.
pH dependence of the histidine resonances of
Zn and Co(II) NS3 protease. a, resonance shift of the
three histidine signals as a function of pH. Zn NS3: His A ( ), His B
( ), His C ( ), and His C+ ( ); Co(II) NS3: His A
( ) and His B ( ). His A and B data points are fitted
with Equation 1, and His C is fitted with a linear equation.
b, His C ( ) peak intensities as a function of pH. Data
were fitted either with Equation 1, assuming a simple equilibrium
(dashed line), or with Equation 2, taking into account
cooperativity (solid line).
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|
We next investigated the pH dependence of the histidine resonances in
the zinc-containing protease. The three
1H signals exhibit a
reversible pH profile (Fig. 3), with peaks A and B shifting downfield
(Fig. 3a) as pH moves from basic to acidic. These signals therefore correspond to a protonation equilibrium, which is in fast
exchange regime on the NMR time scale. The chemical shifts of peaks A
and B were fitted using Equation 1, and the pK values obtained were 6.8 (peak A) and 6.2 (peak B). The pK of 6.8 compares well to the value of
6.9 found in activity titration
experiments2 and thus reflects
the pK of the histidine residue of the catalytic triad
(His57). His B has a pK of 6.2, which reflects the pK of a
protein imidazole moiety exposed to the solvent (33), and was thus
assigned to His110. Peak C (7.84 ppm) behaves differently
(Fig. 3b): in the pH range 8.3-6.2, the chemical shift and
the intensity of the peak is invariant. When the pH was lowered below
6.2, its intensity decreased, and a new peak arose at 8.71 ppm. The pK
of this reversible transition has been estimated at 5.9. The best data
fit was achieved by Equation 2, which yields a Hill coefficient of
n = 2.1 ± 0.2 (Fig. 3b), indicating a
cooperative association of at least two protons. We could exclude the
possibility that this steep titration curve resulted from irreversible
processes because in the same pH range, the titration curves of the
other two histidine residues could be best described by a one-proton
association, and the same titration curves were obtained proceeding
either from high or from low pH. The line widths of species C and
C+ are 13 and 6 Hz, respectively. The line width analysis
carried out on peak C, assuming the minimal model reaction described by Equation 3, resulted in a k
1 of ~9.4
s
1.
The protonated and unprotonated forms of C are in slow chemical
exchange, as demonstrated by the two-dimensional NOESY experiments (Fig. 4a). The kinetic analysis of
the slow exchange process was carried out assuming the model described
in Equation 3, at pH 6.0. After the fitting procedure (Fig.
4b), we obtained the following macroscopic rates:
k+1 and k
1 values of
5.3 ± 0.9 and 9.1 ± 3.1 s
1 and
RC and RC+
values of 0.5 ± 0.4 and 7.3 ± 3.5 s
1,
respectively.

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Fig. 4.
Magnetization exchange of His149
species. a, two-dimensional NMR NOESY spectra of the
aromatic region of Zn NS3 protease. The spectrum was recorded in
2H2O buffer containing 4%
2H5-glycerol, 0.1% CHAPS, 1 mM
DTT, 10 mM sodium phosphate, pH 6.0, with a mixing time of
100 ms. The spin system C in slow exchange (7.8 ppm) and the protonated
C+ (8.71 ppm) are highlighted with letters and
dashed lines. Peak C was identified with His149.
b, time development of magnetization of peak C. The
intensity values measured for the diagonal ( ) and the cross-peak
( ) of the C-C+ process in the series of two-dimensional
NOESY experiments were fitted simultaneously to the system of Equation 5 with a nonlinear least squares optimization routine, using the
program MLAB.
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In the 15N filtered NOESY and ROESY (34) experiments, all
three His
1H resonances exhibit NOEs, with a hydrogen resonating at
the water frequency (not shown). Because the cross-peak disappears when
the NOESY spectra are acquired in D2O, we can exclude that the frequency observed arises from an aliphatic proton of the protein.
However, it is still difficult to assign it to an NOE arising from
water because, as was pointed out by Otting and Wüthrich (35), in
order to assign an NOE to an interaction between an OH-hydroxyl and a
protein hydrogen, one must rule out the possibility that the observed
effect arises from chemically exchanging protons of the protein itself.
In our case, an NOE involving a hydroxyl hydrogen and the
1H proton
is ambiguous because within less than 3 Å, there are the exchangeable
protons of the
2N and
1N nitrogens.
It is convenient to use the nomenclature introduced by Witanowski
et al. (36) to classify the protonation and the tautomeric states of His residues. In this notation, each nitrogen of the histidine imidazole ring is assigned to one of the following three categories:
if it is an N-H,
+ if it is an N-H of a positively charged ring, and
if it is a nonprotonated N. The reference values
reported in Table I for the different states
of the His nitrogens and for their hydrogen bonding state originate
from several different systematic studies on model systems and on
proteins (33, 36-45). On the other hand, there are very few literature data on the chemical shifts of histidine nitrogen atoms involved in
zinc coordination (45-47), and so far, no attempt has been made to
rationalize the observed values. It is possible for a His residue to
assess both the tautomeric and the protonation state, at a given pH,
from the disposition of the peaks in an 1H-15N
HSQC-type experiment (44, 45). In Fig. 5, the
HSQC at pH 6.6 is shown. At this pH value, we find the following
chemical shifts: His57
2N = 184.0 ppm,
1N = 193.9 ppm; His110
2N = 223.6 ppm,
1N = 169.9 ppm; and His149
2N = 211.9 ppm,
1N = 180.0 ppm. The chemical shift of His57 and
His110 can be explained by an equilibrium between the
cationic and the amphionic forms with equivalent populations for
His57 and about 30% of cationic form for
His110. The chemical shift of His149 deserves
more attention, and its interpretation is given under "Discussion."

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Fig. 5.
15N HSQC. A portion of the
1H-15N HSQC spectra of the NS3 protease domain
at pH 6.6. The cross-peak pattern and the assignment of peaks is
evidenced for all the three histidine residues. The delay during which
1H and 15N signals become antiphase was set to
22 ms (1/(2JNH)) to refocus single-bond correlations. The
schematic diagram of the nomenclature used through the article is shown
at the bottom of the figure.
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Characterization of the H149A Mutant--
In order to assess the
functional role of His149 in zinc coordination, we have
mutagenized this residue into alanine by polymerase chain reaction
site-directed mutagenesis (H149A). The H149A mutation has been already
described as leading to a less active enzyme, which is accumulated in
the insoluble fraction when expressed in bacteria (14, 15).
The H149A mutant was obtained pure to >90% by purification in a
metal-free form from the insoluble fraction (see "Materials and
Methods"). The amount of refolded H149A mutant enzyme was estimated
by active site titration. The number of active sites for H149A was
found to be 60% (±2%) of the total protein. Under the same
conditions, 95% (±5%) of the wild type protein was refolded into an
enzymatically active species. The specific activity of the refolded
wild type protein was found to be indistinguishable from that of a
preparation purified under native conditions.
The H149A mutant was found to have both impaired proteolytic activity,
resulting in decreased kcat values, and an
impaired affinity for the NS4A cofactor peptide (Table
II). To establish the stability of the
zinc coordination in the H149A mutant, we performed an EDTA titration.
EDTA has previously been shown to inhibit Zn NS3 protease activity at
high concentrations (13). In order to rule out differences due to the
refolding protocol, we also compared the susceptibility to EDTA of the
refolded and the native wild type proteins. The mutated protein was
slightly more susceptible to inhibition by EDTA: we determined an
IC50 of 10.1 ± 3.4 mM for the H149A
mutant compared with 21.4 ± 4 mM for the wild type
enzyme (not shown). No measurable difference in susceptibility to EDTA
was observed for the two wild type enzyme preparations (refolded and
native). The relatively small difference in the inhibitory potency of
EDTA between the H149A and the wild type enzymes suggests that the zinc
atom is still strongly coordinated, even in absence of His 149. Therefore, this residue does not seem to play a pivotal role in the
stabilization of coordination once the zinc ion is bound.
Refolding of acid-denatured NS3 protease has been shown to be a
zinc-dependent process, accompanied by a decrease in
tryptophan fluorescence.3 We took
advantage of these findings to determine the role of His149
in the incorporation of zinc into the metal binding site. The H149A and
wild type refolding kinetics were performed at plateau of zinc
concentration with respect to the refolding efficiencies (Fig.
6).

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Fig. 6.
Zinc-dependent refolding kinetics
of acid-denatured apo NS3 wild type and H149A. The fluorescence
(340 nm) traces versus the time of wild type (WT)
and H149A apo protein diluted 1:100 (200 nM) in a refolding
buffer containing 50 mM Tris, pH 7.5, 2% CHAPS, 50%
glycerol, 10 mM DTT to which 10 µM
ZnCl2 or 100 µM EDTA were added. The data in
presence of EDTA were obtained using the H149A protein. The wild type
protein gave the same results.
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The fluorescence trace in the presence of EDTA shows a linear decrease
in time both for the wild type and for the mutant protein. This could
be ascribed to the formation, in the absence of accessible zinc ions,
of a misfolded inactive enzyme. In fact, the resulting proteins were
shown to be devoid of any appreciable catalytic activity.
In the presence of zinc, the wild type and the H149A mutant protein
refold with significantly different velocities (Fig. 6). Furthermore,
the refolding process led to only 60% of recovery of total active
sites for the H149A mutant, whereas 95% of active enzyme molecules
were recovered upon refolding of the wild type enzyme. These findings
suggest that the H149A refolding kinetics could be ascribed to the sum
of two competing processes, one that drives the formation of an active
protein and a second one that leads to a misfolded, inactive enzyme.
Increasing zinc concentrations did not augment the amount of refolded
mutant protein, indicating that the mutation does not affect metal
binding itself but rather leads to an incapacity to promptly assume an
active conformation upon interaction with zinc ions.
Perturbation of the Metal Coordination Sphere by NS3
Ligands--
We next addressed the question of whether conformational
changes may occur in the metal coordination sphere. To this purpose, the effects of complex formation with the NS4A cofactor peptide and
with a substrate peptide have been studied. Both substrate and
co-factor binding have been shown to be glycerol-dependent (11). This precluded observation by NMR due to the excessive line-broadening caused by the high viscosity of glycerol-containing buffer solutions. We therefore chose to introduce Co(II) into the metal
binding site, taking advantage of the properties of Co(II) as a
spectroscopic probe. The high structural similarity between the Co(II)
and the Zn NS3 is highlighted by the identical position in the NMR
spectrum, recorded at a low glycerol concentration, of the His signals
A and B, which also show the same pH dependence in the two proteins, as
well as by the similar far UV circular dichroism spectra (data not
shown). This evidence and the comparable kinetic data (Table II) let us
consider the Co(II)-NS3 as a good structural substitute of the wild
type zinc protein.
UV/Visible spectroscopy and circular dichroism were chosen as
techniques, because the Co(II)-substituted protein failed to yield good
quality low-temperature electron paramagnetic resonance spectra,
possibly due to fast relaxation rates and resulting line broadening
(data not shown).
Co(II) NS3 shows complex ligand field spectra with two major bands at
640 (
= 460 M cm
1) and 685 nm (
= 400 M cm
1) and two minor shoulders on either side
at 585 and 740 nm. This transition envelope is characteristic of
distorted tetracoordinated high-spin Co(II) complexes (28, 29). A
strong charge transfer S
Co(II) band, belonging to the three Cys
residues in coordination, was observed at 365 nm with a shoulder at 320 nm. Changes in the Co(II) ligand field spectrum became evident at pH
values below 6, consistent with a change in the coordination geometry
upon protonation of His149 (not shown). At this acidic pH,
the Co(II) NS3 achieved an unstable conformation, which is particularly
prone to losing the metal ion. Therefore, in order to test the effect
of protonation of His149 on the affinity of the enzyme for
the NS4A cofactor, the dissociation constants for the Zn NS3-NS4A
peptide complex were determined at pH 7.5 and 5.1. We obtained values
of Kd = 5.3 µM (pH 7.5) and
Kd = 70 µM (pH 5.1), suggesting that
the presence of His149 in the metal coordination sphere is
required to allow efficient co-factor binding. This loss in complex
stability is comparable with the one observed for the H149A mutant
protease (at pH 7.5 Kd = 41.3 µM),
when the metal is no longer ligated by the imidazoyl moiety.
Upon addition of the NS4A peptide, we recorded the following changes in
the optical spectra: in the envelope of the d-d transitions, the 685 nm
band decreased, whereas the shoulder at 740 nm became more intense
(Fig. 7a). Furthermore, the
intensity of the charge transfer bands decreased (data not shown) and
the intensity in the protein aromatic UV region, around 280 nm,
increased (data not shown). This latter effect on the protein UV
absorbance is not a specific rearrangement of the Co(II) protein
because we also observed it with the zinc protein. These changes in the
spectroscopic properties of Co(II) NS3 all reached a plateau at a 1:1
stoichiometric ratio of protein: cofactor (Fig. 7b).

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Fig. 7.
Perturbation of the Co(II) NS3 ligand field
transitions by the NS4A peptide. a, to 80 µM Co(II) NS3, increasing amounts of NS4A peptide were
added in 50% glycerol, 2% CHAPS, 30 mM NaCl, and 20 mM sodium phosphate, pH 7.5, and electronic absorption
spectra were recorded. Arrows indicate the direction of the
intensity changes upon cofactor binding. b, the differences
in the extinction coefficient (mM) at 690 nm are plotted
versus the protein cofactor equivalents. Data from three
different experiments are shown.
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The CD spectrum of the region between 300-420 nm (Fig.
8a) shows a positive band at 365 nm that matches with an intense peak in the absorption spectrum and a
negative shoulder at 350 nm that is followed by a negative band with
the minimum at 322 nm. This latter band corresponds to a shoulder
recorded in the absorption spectrum. These bands can be assigned to a
complex S
Co(II) charge transfer system, because the number of
expected transitions (S
;
dx2,
dx2-y2) for three cysteines is 12 (49). Upon
binding of the NS4A peptide to Co(II) NS3, the three transitions in the
CD spectrum underwent a decrease in molar ellipticity, reaching a
plateau at a protein:cofactor ratio of 1:1 (Fig. 8b).
Therefore, S-Co(II) bonds are structurally reorganized upon binding of
the peptide cofactor.

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Fig. 8.
Circular dichroism spectra of the Co(II) NS3
S Co charge transfers upon cofactor binding. a, 40 µM Co(II) NS3 was incubated with increasing amounts of
NS4A peptide in 50% glycerol, 2% CHAPS, 30 mM NaCl, and
20 mM sodium phosphate, pH 7.5, and CD spectra were
recorded. b, the differences in molar ellipticities at 322 nm ( ) and 374 nm ( ) are plotted as a function of added NS4A
peptide equivalents.
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The reported changes in both CD and visible spectra are indicative of
rearrangements in the metal coordination geometry occurring upon
formation of the NS3-NS4A peptide complex. We next explored whether
conformational changes affecting the metal binding site also occur upon
substrate binding. To this purpose, we used a Co(II)-substituted
catalytically inactive mutant protein in which the active site serine
residue was mutagenized into alanine (S139A). The protein showed
1H-15N NMR spectra that were virtually
superimposable on those obtained with the wild type enzyme (not shown).
Furthermore, visible and CD spectra and their perturbation by the
addition of the NS4A peptide were indistinguishable from those of the
wild type Co(II) protein. Addition of a substrate peptide harboring the
sequence of the polyprotein NS4A/NS4B junction to S139A Co(II)-NS3 only resulted in a very small change of the ligand field spectrum
characterized by a slight decrease of the two major bands at 640 and
685 nm (data not shown). Furthermore, addition of NS4A peptide to
samples at substrate saturation gave rise to the same spectroscopic
transitions recorded for the enzyme in the absence of substrate (data
not shown). Thus, we can conclude that the substrate does not
significantly rearrange the metal coordination geometry, whereas in the
ternary complex, the metal experiences a structural environment similar to that in the binary enzyme-cofactor complex.
Activation of NS3 Protease by Metal Ligands--
Because we have
shown that the NS3 protease activator NS4A causes a conformational
rearrangement of the metal binding site, we wanted to test whether
externally added metal ligands that perturb the metal coordination were
also capable of modulating the enzymatic activity. As a proof of this
principle, we chose CN
, due to its high affinity for both
zinc and cobalt (49), as well as N3
. Addition of KCN
or NaN3 to Co(II)-substituted NS3 protease caused dramatic changes in
the visible spectrum of the ligand field (Fig. 9). These changes titrated with a
Kd of about 7 mM for N3
and with a Kd = 106 ± 51 µM for CN
. Both N3
and
CN
also enhanced Co(II) NS3 activity by a maximum of
1.5-fold. The CN
activation of the Co(II)-substituted
enzyme titrated with Kd = 176 ± 96 µM, which is in good agreement with the
Kd value obtained from the titration of the visible
spectral bands (data not shown). CN
activation was also
observed with the native, zinc-containing protein. In this case, a
maximum 1.4-fold activation was observed that titrated with a
Kd = 111 ± 51 µM (data not
shown). The activatory effects of CN
and
N3
were specific and not shown by other anions such
as Cl
, I
, or PO43
, which
did not perturb the Co(II) coordination sphere. In fact, both
I
and PO43
were competitive inhibitors
of the enzyme and thus behaved similarly to Cl
, which has
previously been reported to be a competitive inhibitor of the NS3
protease (18).

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Fig. 9.
Perturbation of the Co(II) NS3 ligand field
transitions by cyanide and azide. To 40 µM Co(II)
NS3 and 150 µM 4A peptide in 50% glycerol, 2% CHAPS, 30 mM NaCl, and 20 mM sodium phosphate, pH 7.5 ( ), 0.5 mM KCN ( ) or 50 mM
NaN3 ( ) was added, and spectra were recorded.
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DISCUSSION |
The crystallographic structures (16, 17) of the NS3 protease have
shown that the protein folds in a chymotrypsin-like fold consisting of
two
-barrel-like domains. The topology of these domains is crucial
for the correct orientation of the residues of the catalytic triad,
which are distributed between the domains. Most of the
chymotrypsin-like proteases have disulfide bridges that are believed to
maintain the relative orientations of the residues involved in
catalysis (50). Disulfide bridges present in these extracellular serine
proteases are unlikely to be stable in the reducing intracellular
milieu. In a series of viral proteases, which accomplish their
physiological role intracellularly, such as the NS3 protease domains of
HCV, GB viruses A and B, and hepatits G virus, as well as in the
picornavirus 2A proteases, zinc binding sites probably play an
analogous role of structural stabilization. In contrast to disulfide
bridges, these metal binding sites are stable in the reducing
intracellular milieu. It is remarkable that the conservation of the
three cysteines and of the histidine residue in these enzymes is even
stronger than the one of the catalytic triad because the 2A proteases
belong to the chymotrypsin-like cysteine protease family (13).
In the present work, we have undertaken a spectroscopic investigation
of the metal binding site of the HCV NS3 protease. We tried to asses,
in solution, the coordination role of His149. To address
this issue, we have investigated the NMR spectra of Zn- and
Co(II)-substituted proteins. The nonexchangeable protons of the three
histidine residues were assigned and investigated by their pH
dependence and by the Co(II) -induced isotropic shift of the signals.
The signals arising from His57 and His110 show
a noncooperative pH profile and a peak resonance shift characteristic of a fast exchange process.
The pH dependence profile of His149 has revealed some
peculiarities: it titrates with pK 5.9, lower than the pK of a protein histidine side chain exposed to the bulk water (pK = ~6.2) (33) but still significantly higher than that of a directly coordinating imidazoyl moiety (pK < 5.3) (51). Furthermore, the pH titration profile is consistent with the cooperative association of at least two
protons (Hill coefficient, n = 2.1) in the histidine
protonation process. At acidic pH, the dominant species is expected to
be the protonated His149, corresponding to peak
C+. This species is unlikely to bind the metal ion due to
electrostatic repulsion of the positive charges of the metal and the
protonated His residue. The difference in line width between the
unprotonated and the protonated forms of His149 points
toward a more rigid conformation for the former, whereas most probably,
the latter is freely rotating (52). The experimental kinetic exchange
parameters for the process in equation 3 allow us to calculate a
k
1 = 9.1 ± 3.1 s
1. This
value is similar to one recently published for the conformational switch of the histidines ligating the zinc in the HIV-1 integrase N-terminal protein (53), although in this case both states involved were ordered. Our experimental evidence suggests that the imidazole moiety of His149 modulates the accessibility of the zinc
ion, allowing an "open" and a "closed" conformation in the
protonated and unprotonated states, respectively. This switch mechanism
also parallels the observation in one published crystallographic
structure (16), in which His149 is postulated to
participate in metal coordination in only two of the three molecules in
the asymmetric unit, whereas in the third, the imidazoyl side chain of
His149 moves away.
NOESY and ROESY spectra provided evidence for an NOE involving the
1H nonexchangeable hydrogen and either a water molecule or an
exchangeable group of the protein resonating at water frequency (in
this case, the only candidates are the
2NH and the
1NH of the
same residue). The 15N peak positions in the HSQC
experiment are compatible with the
1N being in a
state and the
2N in an
state (Table I). This tautomeric state is coherent with
what can be inferred from the x-ray structures (16, 17), in which the
nitrogen involved in metal chelation is the
1N, which is therefore
not protonated. In our case, the
state nitrogen corresponds to the
1N and its chemical shift falls in the range described by literature
data (Table I). In contrast, the
state nitrogen (
2NH) is shifted downfield with respect to reference values. This, according to Bachovchin (41), would be an indication of its involvement, as donor,
in a strong hydrogen bond. However, because nitrogen is a very
sensitive nucleus, in proteins, other factors can also alter its
chemical shift to the same extent of an H-bond (44), e. g.
an indirect influence of the involvement of the
1N in metal binding
on the electronic distribution in the coordinated imidazol ring.
The simplest explanation for this experimental evidence is the
assumption that His149 is ligated to the metal using the
1N through an OH
. This model accounts both for the
intermediate pK value and for the stoichiometry necessary to fit the
titration data. In fact, the simultaneous association of two protons is
required to protonate both the histidyl imidazole and the bridging
hydroxyl. We also observed the NOE expected if such an oxhydril group
was actually present. However, we are well aware that by itself this
observation is not unambiguous. In line with crystal structure data,
our spectroscopic findings are compatible with the chelation of zinc
occurring via the
1N. This is not the usually preferred situation
for histidine residues, occurring in only 2 cases out of the 14 described for which the 15N chemical shifts are available
(46-48).
An alternative model has to invoke the presence of a hydrogen bond
acceptor in the vicinity of the
2NH, the pK of which, by chance,
should match that of His149. In fact, only a cooperative
protonation of the
1N and of the hydrogen bond acceptor group could
justify the Hill coefficient of the pH titrations. The binding to zinc
in this case should occur directly with the
1N. However, this second
hypothesis appears less plausible. In fact, to account for the observed
NOE we should admit an exchange process of the
2NH with the bulk
water. But if this hydrogen position is assumed to be in fast exchange
with water (to the extent of not being directly observable) it is
unlikely to be, at the same time, involved in an interaction with a
hydrogen bond acceptor group of the enzyme. Another objection comes
from the crystallographic structures. In fact, it is well established that the zinc binding site is on the surface of the protein and that in
the vicinity of His149 there are no candidate groups that
could act as hydrogen bond acceptors or could be protonated with a pK
reasonably close to the observed one (the carbonyl groups of the
peptidic bonds are the only groups below 5 Å distance that could be
involved).
It is presently not clear whether the zinc ion present in NS3 is the
same zinc ion shown to be essential for the NS2-NS3 autoprotease activity. If so, it could be speculated that a facile movement of
His149, leading to exposure of the coordinated metal, might
play some role in the so far not completely understood mechanism of
proteolysis of the NS2-NS3 junction. In this context, it is interesting
to notice that the sequence pattern of the metal binding residues of
the picornavirus 2A proteases
(Cys-X-Cys/Cys-X-His) differs from the pattern of
HCV-related viruses (Cys-X-Cys/Cys-X-X-X-His). The insertion of two additional residues between Cys145 and
His149 may well contribute to the observed conformational
flexibility of His149 and could be related to a putative
role of the metal, in HCV-related viruses, in the processing of the
NS2-NS3 site.
The differences in the spacing of the metal-coordinating residues in
HCV NS3 and in picornavirus 2A proteases could also explain the
different effects of mutagenesis of His149 and its
corresponding residue in 2A. In fact, biochemical characterization of
the H149A mutation in the NS3 protease (14, 15) has pointed out that
the removal of the imidazoyl moiety only leads to minor effects on
enzyme activity but causes the protein to accumulate in the insoluble
fraction when expressed in E. coli (15). On the other hand,
mutagenesis studies on 2A proteases from rhinovirus (53) and poliovirus
(54) have revealed the essential role of the residue corresponding to
His149 of HCV NS3 for the catalytic activity of these
enzymes. These results, indeed, are rather qualitative because they
have been obtained in in vitro translation experiments. We
therefore decided to quantitatively characterize the purified H149A
mutant protein. Our results indicate that the H149A mutation has a
major impact on the productive incorporation of zinc ions into the
metal binding site during folding of the protein. In the mutant, this
process was shown to occur on a time scale similar to misfolding,
leading to only partial recovery of active protein. In contrast, only a
2-fold-increased susceptibility to EDTA-inactivation was observed, suggesting that, once incorporated, the metal is bound with a similar
affinity in wild type and H149A mutant proteins. The mutation further
affected both specific activity and affinity of the protein for its
co-factor. Experimental difficulties did not allow the introduction of
Co(II) into the metal binding site or concentration of the mutant
protein, thus impeding a spectroscopic characterization of the
conformational consequences of the H149A mutation. The activity data,
however, suggest that changes in the coordination geometry resulting
from the H149A mutation may have significant effects on the protein
structure.
The relationship between metal coordination geometry and enzymatic
activity is further highlighted by the spectral changes that accompany
the complexation of the NS3 protease with its NS4A co-factor peptide.
In fact, electronic absorption and circular dichroism spectra
demonstrated ligand-metal rearrangements of the Co(II)-substituted NS3
protease induced by binding of the co-factor, whereas no significant
effects could be detected upon substrate binding to the active site.
Movements of cysteine ligands seemed to play a crucial role in
rearrangements of the coordination geometry, as pointed out by the
changes in the circular dichroism of the S
Co(II) transitions.
Activation of the HCV NS3 protease by its co-factor NS4A involves both
a structural reorganization of the N-terminal domain and a
rearrangement of the catalytic triad (16, 17). Our data show that at
least some of these conformational transitions involve also
rearrangements of the metal binding site. In fact, it may be argued
that the metal coordination sphere is endowed with an intrinsic
flexibility, necessary to respond to the conformational rearrangements
of the protease during its complexation with the NS4A cofactor. Indeed,
His149 seems to play a major role in this conformational
flexibility, as in the open metal coordination, where the
His149 is not ligating, and in the H149A mutant protease,
the NS3-NS4A complex stability is reduced.
NS4A binds to the N-terminal region of NS3 (55). Interestingly, Mori
et al. have shown that deletions in this region lead to an
enhanced susceptibility toward inactivation of the NS3 protease by EDTA
(56), which is suggestive of an NS4A-dependent tightening of metal-ligand bonds and in line with our findings of cofactor-induced conformational changes of the metal coordination.
The relationship between coordination geometry and enzymatic activity
suggests that compounds having the capability of perturbing the native
zinc coordination in the NS3 protease might modulate the enzymatic
activity. We have shown, as a proof of principle, that CN
has such an effect. CN
was shown to bind to the metal in
the Co(II)-substituted enzyme, inducing spectroscopically detectable
changes in the coordination geometry that titrated with the same
apparent Kd as the activation process. Furthermore,
CN
was able to enhance also the activity of the native,
zinc-containing protease.
Perturbations of the ligand field spectrum of Co(II) substituted
proteins by externally provided ligands have been used to probe the
presence of transient coordination moieties such as water molecules
(57, 58). Our observations on CN
and N3
effects are in line with these reports, suggesting a distorted tetrahedral site, at which the water ligand has been replaced by the
added anions.
In the light of the described correlation between metal coordination
geometry and enzymatic activity, it is tempting to speculate that
compounds capable of perturbing the metal coordination geometry of the
NS3 protease might either activate or inhibit the enzyme, depending on
the nature of the induced perturbation, thus revealing a new potential
mechanism for NS3 protease inhibitors.
We thank Prof. Alessandro Desideri and Dr.
Francesca Polizio for helpful discussions and for performing electron
paramagnetic resonance experiments. We are particularly grateful to Dr.
Angelo De Martino for his expert advice in atomic absorption
spectroscopy. We thank Dr. J. G. Omichinski for the useful
clarifications on the fitting procedures and Dr. Anna Tramontano for
critical reading of the manuscript. Finally, very special thanks go to
Dr. Michael Geeves and all the organizers of the Transient Kinetics
Applied to Biological Macromolecules EMBO course (1997) for their
invaluable suggestions.