From the
Departments of Biochemistry and Molecular
Biology and **Ophthalmology and Visual Sciences and
the ¶Department of Medicine and James Graham
Brown Cancer Center, University of Louisville School of Medicine, Louisville,
Kentucky 40292
Received for publication, December 12, 2002 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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Alkaline proteinase (APR),1 the serralysin homolog of Pseudomonas aeruginosa, was first described in 1963 by Morihara (2). Subsequent x-ray crystallography of APR (3, 4) and Serratia marcescens metalloproteinase (5) revealed that these metalloproteinases consist of two domains, an N-terminal catalytic domain of about 230 residues and a C-terminal domain of similar size that is required for secretion of the enzyme across the periplasm into the external medium (6).
The function of the serralysin inhibitors is probably to protect the
bacterium from adventitious proteolysis during serralysin secretion
(7). They are produced as
precursors of about 125 amino acids with an N-terminal signal sequence that is
removed to form the mature inhibitor of 103106 residues
(8). The seven serralysin
inhibitors known to date share 20% sequence identity and 4050%
sequence similarity (9). In
particular, residue 2 is always Ser, whereas residue 3 is Leu in 6 of 7 known
inhibitors and Phe in the seventh. Position 4 exhibits greater variation and
can be either non-polar (Leu or Val) or polar (Arg or Lys).
Crystal structures of the S. marcescens serralysin-Erwinia
chrysanthemi inhibitor Inh and APR·APRin complex from P.
aeruginosa have been determined
(9,
10). These structures show
that both the Erwinia and the Pseudomonas inhibitors fold
into disulfide-stabilized, eight-stranded -barrels with an N-terminal
trunk of 10 amino acids with the first five residues of the trunk occupying
the extended substrate binding site of the enzyme (see
Fig. 1).
Ser-1I,2 which is
conserved in all known serralysin inhibitors except S. marcescens
(where the N terminus is Gly), straddles the S1-S1' substrate binding
pockets of the enzyme, which allows the
-amino and carbonyl groups of
the N terminus to chelate the catalytic zinc. The hydroxyl proton forms a
hydrogen bond with the hydroxyl oxygen of Tyr-216P and the hydroxyl group of
Ser-2I donates a hydrogen bond to the carboxyl of the catalytic base,
Glu-177P. The
-barrel, which makes several contacts with the enzyme near
the Met turn, prevents the trunk from extending past the zinc and completely
occupying the extended substrate binding site
(10).
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Characterization of the interaction between APR and its inhibitor is of interest for at least three reasons. First, APR is one of several virulence factors of P. aeruginosa capable of inactivating proteins involved in host defense mechanisms (5, 11, 12); understanding inhibition of this enzyme could result in strategies for the amelioration of infections by this organism. Second, the activity of both APR and the structurally similar mammalian MMPs is regulated by the formation of high affinity complexes with specific protein inhibitors. Finally, the mechanism of interaction between serralysins and their inhibitors is similar to that of the MMPs and their cognate inhibitors, the TIMPs (9), although the two classes of inhibitor bear no apparent sequence homology. In both the N-terminal residue of the inhibitor chelates the catalytic zinc; in addition, both inhibitors have a hydroxyl group in the penultimate position that hydrogen bonds to the catalytic Glu of the enzyme (9, 13, 14).
The goal of this study was to investigate the role of side chains in positions 25 in the interaction between APRin and APR. We found that single amino acid changes at these positions resulted in altered binding affinity, with conserved Ser-2I more sensitive to mutation than positions 35, in keeping with its specific interactions with the proteinase. Molecular dynamics simulations were used to rationalize these alterations in affinity in structural terms.
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EXPERIMENTAL PROCEDURES |
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Protein Expression and PurificationRecombinant proteins
were expressed in Escherichia coli strain BL21(DE3) obtained from
Novagen. The procedures of Feltzer et al.
(15) for culture, protein
induction, osmotic shock, and inhibitor purification were followed except that
a broad-spectrum proteinase inhibitor mixture designed for bacterial cell
extracts (Sigma) was included in the crude osmotic lysate. The inhibitor
proteins were purified by ion exchange and gel filtration chromatography and
subsequently characterized by SDS-PAGE, electrospray mass spectrometry,
fluorescence emission, and circular dichroism spectroscopy. Each mutant gave a
single band on SDS-PAGE and the expected molecular mass by mass spectrometry.
For all inhibitors except those with an added Trp residue, protein
concentrations were estimated from the absorbance at 280 nm using =
17.7 mM1
cm1 previously established for the wt protein
(15). An
280
of 25.3 mM1 cm-1 was
estimated for the S2W and I4W mutants using the method of Gill and von Hipple
(16).
Alkaline ProteinaseCrystalline APR was purchased from
Nagase Biochemicals Ltd., Tokyo, Japan, and purified as described
(15). APR concentration was
estimated by UV absorption using
at 280 nm, in
conjunction with a molecular weight of 50,000
(2).
Kinetic AssaysAPR inhibition was assessed as previously
described (15) using
Ac-Pro-Leu-Gly-[2-mercapto-4-methyl-pentanoyl]-Leu-Gly ethyl ester as
substrate (17). The kinetics
of substrate hydrolysis were determined by measuring the appearance of the
product peptide
HSCH2CH[CH2CH(CH3)2]-Gly-OEt
spectrophotometrically using the thiol indicator 4,4'-dithiodipyridine
(18) at a concentration of 0.5
mM. Reactions were conducted in microtiter plates that had been
pretreated with a siliconizing agent (Sigmacote) to minimize protein binding
to the plastic. Buffer conditions were 50 mM MOPS, 5 mM
CaCl2, 0.001% bovine serum albumin, pH 7.0, with either 0.1 or 2.4
M NaCl. The progress curves were determined with a Spectramax
microtiter plate reader (Molecular Devices, Sunnyvale, CA) set to 324 nm and
maintained at 25 °C. In a typical set of assays, a total of 8 reactions at
inhibitor concentrations ranging from 0 to 5 nM were monitored
at 20-s intervals for a period of 4 h. The concentration of APR was usually
0.5 nM, except for an enzyme-free blank that was included to
correct for spontaneous hydrolysis of the thioester substrate.
Determination of Kinetic and Equilibrium ConstantsInhibition of APR by APRin in this assay system has been previously shown (15) to be compatible with the reaction steps defined in Scheme 1.
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The dissociation constant Ks for the ES complex and kcat for ES were set to values determined previously (15). Values for rate constants for enzyme-inhibitor association and dissociation (kon and koff) and for the rate constant for spontaneous hydrolysis of the substrate (kh) were estimated by global fitting of a series of progress curves obtained at different E:I ratios to the kinetic equations of Scheme 1 using the computer program Dynafit (15, 19). The kinetic constants reported are the mean of at least three independent sets of experiments, each of which covered a range of inhibitor concentrations. For some inhibitors, koff was too small to be reliably determined by this procedure. In these instances it was determined directly by dilution of the pre-formed enzyme-inhibitor complex into an assay mixture followed by recording the appearance of active enzyme accompanying relaxation to the new equilibrium state (15).
Fluorescence and CD SpectroscopyFluorescence and CD
spectroscopy were used to assess the effects of the mutations on gross protein
structure. Fluorescence emission spectra were recorded at room temperature
from 290 to 460 nm using a PerkinElmer Life Sciences LS50B spectrofluorometer.
The excitation wavelength was 280 nm, and the protein concentration was 0.5
µM in 20 mM Tris, 50 mM NaCl, pH 7.4, or
20 mM Tris, 6 M guanidine HCl, pH 7.6. CD spectra were
measured at room temperature with a Jasco J-710 spectropolarimeter calibrated
with d-10-camphor sulfonic acid. The path length was 0.02 cm, and the
protein concentration was 2040 µM in 20 mM
Tris, 0.1 M NaCl, pH 7.4. Four ellipticity readings at 1-nm
intervals between 240 and 187 nm were averaged and converted to
values based upon the mean residue weight of each protein. The program CDsstr
(20) was used to estimate
secondary structure from the CD data. All fluorescence emission spectra and CD
spectra were corrected by subtraction of a buffer blank.
ModelingStarting models were based on the APR·APRin x-ray crystal structure (9) with the inclusion of residues 26251 catalytic domain of the enzyme. These residues encompass all those in contact with the inhibitor in the crystal structure (see Fig. 1). The inhibitor, zinc atom, and 58 water molecules in the interface between APR and APRin were included from the x-ray crystal structure. The S2D and I4W mutant inhibitors were created by replacing the corresponding inhibitor residues with mutated residues in standard conformations. Models were hydrated in a 10-Å box of TIP3P waters using standard AMBER (21) rules; Na+ counterions were placed randomly for charge neutrality. Box sizes were adjusted to include 10,398 waters for each model. Simulations were performed in the isothermal isobaric ensemble (300 K, 1 atm) with the AMBER 7.0 program (21) and parameters from parm96.dat using periodic boundary conditions and the Particle-Mesh-Ewald algorithm. Molecular dynamics simulations used the MPI version of the Sander routine (1.5-fs time step) with SHAKE to freeze all bonds involving hydrogen. Initial equilibrium for 155 ps following the general protocol of Trent (22) was performed with gradual removal of positional restraints on the protein complex. The production runs were 1.5 ns in length, and average structures for each complex (taken from 50 snapshots accumulated in the last 75 ps) were obtained and subsequently minimized. The calculations were run on a 32 processor SGI Origin 2000.
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RESULTS |
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The CD spectra of the mutant proteins in the spectral range 190240 nm was used to assess secondary structure. The spectra of all the mutants were virtually superimposable with the spectrum of the wt protein (data not shown), indicating similar secondary structures. In keeping with the spectral similarity, analysis using the CDsstr program (20) showed that the secondary structure of the mutants did not differ significantly from that of wt APRin. We conclude from the fluorescence and CD experiments that the mutant proteins fold into similar conformations that resemble that of the wt protein.
Kinetic StudiesThe development of inhibition of APR by wt APRin required 34 h to reach completion at the nM concentrations of the proteins used in the assays (15). Similar time-dependent inhibition kinetics was displayed by the mutants in this study (data not shown). As demonstrated in our previous work (15), the mechanism of inhibition is consistent with a competitive, single-step bimolecular reaction between enzyme and inhibitor as depicted in Scheme 1.
The rate constants for formation and breakdown of the APR·APRin complex along with values for the apparent dissociation constant and change in Gibbs free energy of binding derived from these constants are summarized in Table II. Of the 11 APRin mutants, 10 exhibited decreased affinity for APR. One, the I4W mutant, showed an increased affinity for APR compared with the wt protein.
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Ser-2I was most sensitive to mutations of the positions examined.
Alteration of Ser-2I to Asp, Arg, or Trp resulted in decreased
enzyme-inhibitor affinity by 35-, 180-, and 14-fold, respectively. For each
mutant in this series, the decrease in binding affinity resulted predominantly
from an increase in the rate of dissociation, with elevations of 35-,
375-, and 60-fold in koff for the S2D, S2R, and S2W
mutants, respectively. The Arg and Trp mutants exhibited an increased
association rate of
2- and
5-fold, respectively, whereas the rate of
association of the Asp mutant was about the same as that for the wt
protein.
Inhibitor positions 3, 4, and 5 were less sensitive than position 2 to
alteration. A decrease in affinity of 1015-fold was observed for these
mutants except for I4W, which exhibited a 3-fold increase. For mutant L3E, the
change in affinity was comparable with that for S2W (both 10-fold),
whereas mutation at positions 4 or 5 resulted in smaller changes
(<10-fold). As with position 2, the affinity changes resulted predominantly
from an increase in rate of dissociation. The higher affinity observed for the
I4W protein resulted completely from an increase in the rate of
association.
Ionic Strength EffectsInhibition assays were routinely
conducted in the presence of 2.4 M NaCl because the sensitivity of
the assay is better under these conditions as a result of a 6.4-fold decrease
in Ks at higher the ionic strength
(15). To ensure that high
ionic strength did not mask potential electrostatic effects associated with
binding of the S2D, S2R, I4E, I4R, and L5E mutants, we also tested each of the
charge-altered mutants at lower ionic strength (0.1 M). The
decrease in salt concentration resulted in a maximum increase of 5-fold
in kon, which was similar to that previously observed for
wt inhibitor (15). This
relatively small effect of ionic strength on kon suggests
that the binding interactions are not strongly driven by electrostatic
interactions.
ModelingTo provide a structural basis for the observed alterations in binding affinity, we conducted a molecular dynamics simulation of the binding of two inhibitors, S2D and I4W, to the catalytic domain of APR. In addition, we simulated binding of the wt inhibitor to ensure that we could produce a structure in reasonable agreement with the crystal structure of the complex between APRin and APR. Because of the computational resources required for these relatively large complexes, it was not practical to carry out a molecular dynamics simulation of all of the complexes with mutant inhibitors.
In all the models, the fold of the enzyme and the geometry of the zinc
binding site were well maintained. The molecular dynamics simulation of the wt
inhibitor maintained a hydrogen bond from Ser-2I to Glu-177P (3.3 Å O to
O) with the hydroxyl hydrogen oriented directly at Glu-177P
(Fig. 2). The hydroxyl oxygen
of Ser-2I also interacted electrostatically with the zinc atom (2.7 Å O
to Zn2+). The N-terminal
in the model structure rotated away
from the zinc and formed a hydrogen bond to the carbonyl oxygen of Ala-135P, a
departure from the wt crystal structure, which shows the unprotonated
N-terminal NH2 group interacting with the catalytic zinc. This
shows that the protonated N terminus can form an alternative, energetically
favorable interaction with the proteinase that does not involve the zinc.
The mutated inhibitors were also stable in the complexes and did not
dissociate from the receptor in the simulation. Molecular dynamics of the S2D
mutant (Fig. 2) revealed that
the aspartic residue is compact and held in place by an electrostatic
interaction with the zinc atom (2.8-Å carboxylic O to
Zn2+) and a hydrogen bond to Leu-3 of the inhibitor
(2.4-Å carboxylic O
to amide backbone NH). The Glu-177P
carboxylate is 3.2 Å from the aspartic carboxylate, which would have a
destabilizing repulsive effect. However, this repulsive effect would be
reduced by partial charge neutralization by the zinc. The N-terminal hydrogen
bond to Ala-135P noted in wt complex is no longer present.
The molecular dynamics of the I4W mutant
(Fig. 3) revealed a major
change in the flexible loop (residues 189200) of the enzyme with
movement of the Glu-194P at the apex of the loop by nearly 9 Å
(measuring P194 C WT to P194 C
I4W, 8.6 Å). The Trp
residue at position 4 stacks over Tyr-158P for favorable
-
interactions with movement of the adjacent loop (residues Ser-130P to
Gly-133P) to close into Trp-4I to form hydrophobic interactions. Ser-2I
maintains an electrostatic interaction with the zinc atom (O to
Zn2+, 2.9 Å); however, the hydroxyl hydrogen is
now oriented directly at the carbonyl oxygen of Ala-135P (H to O 1.9 Å)
rather than toward the carboxyl of Glu-177P.
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DISCUSSION |
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We expected that mutation of Ser-2I to Arg might increase the stability of
the complex by replacing the Ser-2I to Glu-177P hydrogen bond with a
potentially stronger electrostatic interaction. This new interaction could
increase the stability of the complex, possibly manifesting itself as a
decrease in the dissociation rate due to the electrostatic attraction between
the two groups. However, we observed an increase in the rate of complex
formation as well as a larger increase in the rate of dissociation, which
translates into a decrease in stability by 3 kcal/mol
(Table II). Examination of a
model of the mutant complex constructed by simply replacing Ser-2I with Arg
and selecting the optimum side chain conformation without adjusting the
geometry of the binding pocket revealed that the S1' pocket of APR,
which accepts the side chain of Ser-2I, is too small to accommodate the
increased size of the Arg residue. Analysis of this mutant using the program
MOLPROBITY3
(24,
25) revealed severe clashes
between Arg-2I and Thr-173P, His-176P, and Glu-177P. Thus, considerable
rearrangement of the receptor site would be necessary to accommodate the more
bulky side chain of Arg. The rearrangement necessary to accommodate the Arg
residue apparently cost more in binding free energy than could be supplied by
formation of an electrostatic interaction between the guanidinium group of the
mutant residue and the carboxyl group of Glu-177P.
Alteration of Ser-2I to the negatively charged Asp residue was expected to
destabilize the complex as a result of electrostatic repulsion between the
carboxyl of the inhibitor and the carboxyl of Glu-177P. In qualitative
agreement with this expectation, the stability of the complex with the S2D
mutant was decreased by 2 kcal/mol, entirely as a result of an increased
off rate. We examined this mutation in more detail by molecular dynamics to
see if the newly introduced carboxyl might form other stabilizing
interactions. The model showed that Asp-2I was well accommodated within the
receptor site, with its carboxyl group directed toward the catalytic zinc
rather than Glu-177P, forming a new electrostatic interaction with the metal
ion. Evidently, this interaction was insufficient to increase the binding
affinity of the complex.
The rationale for substitution of Trp at position I2 was that the indole
side chain might better occupy the S1' pocket, which is non-polar and
incompletely filled by the hydroxymethyl side chain of Ser-2I in the complex
with the wt inhibitor (9). This
pocket contains several aromatic residues that might interact favorably with
an aromatic group of the inhibitor. However, substitution of Ser-2I with Trp
resulted in a decreased affinity (G =
1.5
kcal/mol). As with the Arg substitution at this position, the association rate
constant was increased for the S2W mutant, but the dissociation rate increased
to a greater extent, leading to an overall decrease in affinity. Analysis with
MOLPROBITY suggested that without undergoing significant rearrangement, the
receptor pocket is too small to accommodate Trp.
It is also of interest that the two mutants with larger side chains at the 2 position (e.g. the S2R and S2W proteins) were bound more rapidly than the wt protein by the enzyme. This is consistent with the results of our earlier experiments with deletion mutants (15) where the rate of interaction between the two proteins was markedly dependent on the surface area of the trunk region of the inhibitor.
Position 3, which is also conserved among serralysin inhibitors
(9), was somewhat more
sensitive to mutation than positions 4 (not conserved) or 5 (conserved). Here,
the decrease in affinity of the L3E mutant was about 15-fold, again
predominantly as a result of an increase in the off rate. In the wt complex,
Leu-3I makes non-polar contacts with Ala-134P, Tyr-169P, and Tyr-216P. The
observed decrease in stability of 1.6 kcal/mol may be attributed to the
unfavorable effect of accommodating a charged side chain within this
hydrophobic site.
Position 4, which is not conserved among the known serralysin inhibitors
(9), was least sensitive to
mutation. Alteration of Ile-4I to Ala, Glu, Arg, or Pro produced at most a
10-fold decrease in affinity, which translates into change in apparent free
energy of binding of 1 kcal/mol or less. Mutation to Trp at position 4,
however, resulted in a small increase in the apparent binding free energy of
0.6 kcal/mol. Examination of this mutation by molecular dynamics provided
a reasonable explanation for the observed increase in affinity. The modeled
complex revealed a major movement of a flexible loop of the proteinase
(residues 189200), enabling formation of a
-stacking interaction
between the aromatic rings of Trp-I4 and Tyr-158P
(Fig. 3).
Mutation of Leu-5I to Glu had little effect on kon but
did increase in koff, resulting in a decrease in binding
free energy of 1.1 kcal/mol. Leu-5I contacts Tyr-158P, Tyr-159P, and
Tyr-230P at the S4' site of APR; changing the non-polar Leu to polar Glu
should destabilize these interactions.
It is of interest to compare the effects of mutation of APRin at position 2
with similar mutational studies with the TIMPs. Residue 2 is conserved among
TIMPs and is either Ser or Thr in the four known TIMP molecules. Ser-2 in
TIMP-2 occupies the S1' site of the MMP, and the side chain forms a
hydrogen bond with the catalytic Glu of the MMPs in similar fashion to the
serralysins and their inhibitors
(9,
13,
14). In a mutational study of
TIMP-2, Butler et al.
(26) found for the S2E mutant
that the apparent inhibition constant increased by 400-fold for binding
to MMP-2, MMP-13, MMP-14 but was increased only 4-fold for binding to MMP-7.
In contrast, an S2K mutant showed less than a 2-fold change in inhibition
constant. These authors concluded that the potential of the new side chain to
form a bond with the catalytic Glu of the enzyme did increase binding
affinity. In addition, analysis of their mutants suggested that the presence
of a larger side chain that could more completely fill the S1' binding
pocket of the MMPs did not increase inhibitor affinity.
Huang et al. (27)
and Meng et al. (28)
found with N-TIMP-1 that substitutions for Thr-2 provided the greatest effect
on inhibition of MMPs, increasing the inhibition constant for MMP-1, MMP-2, or
MMP-3 by as much as 2700-fold for a T2D substitution. Substitution of
non-polar residues incapable of H-bonding to the catalytic glutamate had a
relatively modest effect on inhibition, suggesting that this interaction
contributes little to the stability of the complex.
In conclusion, we have demonstrated in this study that alteration of the conserved Ser residue in the serralysin inhibitor APRin results in the largest effect on enzyme-inhibitor affinity of those residues of the inhibitor that interact with the substrate binding site of the enzyme. A similar sensitivity to mutation at the 2 position has also been noted with inhibition of MMPs by N-TIMP-1 and N-TIMP-2. In addition, we have shown that molecular dynamics simulation of the interaction between a metalloproteinase and its inhibitor can lead to reasonable models that provide a qualitative rationalization in structural terms of changes in enzyme-inhibitor affinity brought about by mutation.
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FOOTNOTES |
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Recipient of a University of Louisville Graduate Fellowship.
|| To whom correspondence may be addressed: Dept. of Medicine, J. Graham Brown Cancer Center, University of Louisville, Louisville, KY 40292. Tel.: 502-852-2194; Fax: 502-852-2195; E-mail: john.trent{at}louisville.edu.
To whom correspondence may be addressed: Dept. of Biochemistry and Molecular
Biology, University of Louisville, Louisville, KY 40292. Tel.: 502-852-5226;
Fax: 502-852-6222; E-mail:
rdgray{at}louisville.edu.
1 The abbreviations used are: APR, alkaline proteinase from P.
aeruginosa (EC 3.4.24.40
[EC]
); APRin, alkaline proteinase inhibitor from
P. aeruginosa; CD, circular dichroism; MMP, matrix metalloproteinase;
MOPS, 3-(N-morpholino)propanesulfonic acid; TIMP, tissue inhibitor of
metalloproteinases; N-TIMP, N-terminal domain of TIMP; wt, wild type.
2 The sequence numbering system of Hege et al.
(9) is followed, where the
suffix "I" designates residues in the inhibitor, and
"P" designates residues in the proteinase, with numbering
beginning with the mature sequence of each protein.
3 This program measures and displays atomic contacts between molecular
surfaces in macromolecules using the atomic coordinates of the structure in
conjunction with a small probe (radius of 0.25 Å) and a contact dot
algorithm (24,
25).
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REFERENCES |
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