Alkaline Proteinase Inhibitor of Pseudomonas aeruginosa

A MUTATIONAL AND MOLECULAR DYNAMICS STUDY OF THE ROLE OF N-TERMINAL RESIDUES IN THE INHIBITION OF PSEUDOMONAS ALKALINE PROTEINASE*

Rhona E. Feltzer {ddagger} §, John O. Trent {ddagger} ¶ || and Robert D. Gray {ddagger} ** {ddagger}{ddagger}

From the Departments of {ddagger}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.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alkaline proteinase inhibitor of Pseudomonas aeruginosa is a 11.5-kDa, high affinity inhibitor of the serralysin class of zinc-dependent proteinases secreted by several Gram-negative bacteria. X-ray crystallography of the proteinase-inhibitor complex reveals that five N-terminal inhibitor residues occupy the extended substrate binding site of the enzyme and that the catalytic zinc is chelated by the {alpha}-amino and carbonyl groups of the N-terminal residue of the inhibitor. In this study, we assessed the effect of alteration of inhibitor residues 2–5 on its affinity for Pseudomonas alkaline proteinase (APR) as derived from the ratio of the dissociation and associate rate constants for formation of the enzyme-inhibitor complex. The largest effect was observed at position Ser-2, which occupies the S1' pocket of the enzyme and donates a hydrogen bond to the carboxyl group of the catalytic Glu-177 of the proteinase. Substitution of Asp, Arg, or Trp at this position increased the dissociation constant KD by 35-, 180-, and 13-fold, respectively. Mutation at positions 3–5 of the trunk also resulted in a reduction in enzyme-inhibitor affinity, with the exception of an I4W mutant, which exhibited a 3-fold increase in affinity. Molecular dynamics simulation of the complex formation between the catalytic domain of APR and the S2D mutant showed that the carboxyl of Asp-2 interacts with the catalytic zinc, thereby partially neutralizing the negative charge that otherwise would clash with the carboxyl group of Glu-177 of APR. Simulation of the interaction between the alkaline proteinase and the I4W mutant revealed a major shift in the loop comprised of residues 189–200 of the enzyme that allowed formation of a stacking interaction between the aromatic rings of Ile-4 of the inhibitor and Tyr-158 of the proteinase. This new interaction could account for the observed increase in enzyme-inhibitor affinity.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria of the genera Pseudomonas, Serratia, and Erwinia secrete homologous 50-kDa proteinases and also produce specific, high affinity inhibitors directed against these proteinases that belong to the serralysin branch of the metzincin metalloproteinase superfamily. The metzincin family also includes the mammalian matrix metalloproteinases, the reprolysins, and the adamalysins (1). In addition to a consensus zinc binding sequence consisting of an HEXXHXXGXXH motif, the metzincins are characterized by a conserved surface methionine residue located near the base of the zinc binding pocket.

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 103–106 residues (8). The seven serralysin inhibitors known to date share ~20% sequence identity and 40–50% 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 {beta}-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 {alpha}-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 {beta}-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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FIG. 1.
Ribbon diagram of the complex between APR and its inhibitor, APRin. Molecular dynamics simulation was conducted with the catalytic domain of APR (residues 25P–251P, shown in purple) and inhibitor residues 1I–105I, shown in yellow). Residues 1P–24P (blue) and 252P–470P (green) that do not interact with the inhibitor were omitted from the simulation. A close-up view of the catalytic region is shown in Fig. 2. The diagram was produced with Insight II (Accelrys) from coordinates in Protein Data Bank entry 1JIW [PDB] (9).

 



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FIG. 2.
Structural comparison of the catalytic site region of the complex of alkaline proteinase with wt APRin and the S2D APRin mutant generated by molecular dynamics simulation. The figure shows catalytic Glu-177P of the wt complex hydrogen-bonded to the hydroxyl of Ser-2I (green side chains, 3.3 Å). Superimposed in orange are the carboxyl side chains of Glu-177P (left) and Asp-2I in the mutant, which is directed toward the catalytic zinc (2.8 Å). The spheres represent the zinc atom. Mutant backbone and zinc are shown in blue, and the corresponding wt structures are in white.

 

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 2–5 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 3–5, in keeping with its specific interactions with the proteinase. Molecular dynamics simulations were used to rationalize these alterations in affinity in structural terms.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of APRin Expression Plasmids—APRin mutants were expressed as described previously (15) using the isopropyl-1-thio-{beta}-D-galactopyranoside-inducible pET22b+ expression system obtained from Novagen (Madison, WI). A cDNA encoding the mutant protein was generated by the polymerase chain reaction with primers containing the desired altered codon and restriction sites for cloning (shown in Table I). The template DNA was EcoRI-digested genomic DNA from P. aeruginosa (strain PA01). The resulting products were ligated into the pT7Blue vector using the "Perfectly Blunt" cloning kit of Novagen. The APRin-encoding fragment was then excised from pT7Blue with BamHI and either MscI or Ecl136II. The DNA was purified by agarose gel electrophoresis and ligated into the MscI/BamHI sites of the expression plasmid. The sequence of both strands of each coding insert was verified using a Beckman CEQ 2000 DNA sequencer.


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TABLE I
PCR primers for construction of expression vectors for wt APRin and APRin mutants

Underlined sequences represent restriction sites for the given enzyme.

 

Protein Expression and Purification—Recombinant 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 {epsilon} = 17.7 mM1 cm1 previously established for the wt protein (15). An {epsilon}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 Proteinase—Crystalline 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 Assays—APR 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 Constants—Inhibition 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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SCHEME 1.
Reaction mechanism for data analysis of the interaction of APRin with APR.

 

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 Spectroscopy—Fluorescence 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 20–40 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 {Delta}{epsilon} 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.

Modeling—Starting models were based on the APR·APRin x-ray crystal structure (9) with the inclusion of residues 26–251 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Spectroscopic Characterization of Recombinant Proteins— Fluorescence and CD spectra of the recombinant proteins were compared with the corresponding spectra for the wt protein to compare folding of the recombinant proteins with that of the wt protein. Fluorescence emission spectra of Trp-containing proteins is an indicator of tertiary structure because the emission maximum of the indole ring is sensitive to the polarity of the local environment (23). For the mutant proteins having the same number of Trp residues as the wt protein, the mean fluorescence emission maximum was at 335.1 ± 1.7 nm compared with the emission maximum for the wt protein of 335 nm. This indicates that the three Trp residues in the mutant proteins experience a microenvironment comparable with that of the same residues in the wt protein. The emission maxima for the S2W and I4W mutants were shifted to 340 and 341 nm, respectively, which is consistent with a structure in which the mutant Trp residues are exposed to water, as expected for the solvent-exposed trunk in the proteinase-free inhibitor. Under denaturing conditions, all of the mutants exhibited fluorescence maxima of 355 ± 1 nm, which is characteristic of Trp in aqueous environment (23).

The CD spectra of the mutant proteins in the spectral range 190–240 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 Studies—The development of inhibition of APR by wt APRin required 3–4 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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TABLE II
Kinetic and equilibrium constants for interaction of APR with wt APRin and APRin mutants

Mean ± S.D. of at least three independent experiments.

 

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 10–15-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 Effects—Inhibition 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.

Modeling—To 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{alpha} to Zn2+) and a hydrogen bond to Leu-3 of the inhibitor (2.4-Å carboxylic O{beta} 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 189–200) of the enzyme with movement of the Glu-194P at the apex of the loop by nearly 9 Å (measuring P194 C{alpha} WT to P194 C{alpha} I4W, 8.6 Å). The Trp residue at position 4 stacks over Tyr-158P for favorable {pi}-{pi} 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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FIG. 3.
Structural comparison of the simulated complex of the catalytic domain of APR with wt APRin and I4W APRin generated by molecular dynamics simulation. The loop comprised of residues 189P–200P in the wt complex (orange ribbon) is shown in green. In the simulated I4W complex (white ribbon), this loop (in red) moves toward Trp-4I. Also illustrated is the proximity of Trp-4I (purple) and Tyr-158P (blue), which promotes {pi}-stacking interactions between the aromatic rings.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The goal of the current study was to assess the role of inhibitor residues 2 through 5 in complex formation between APRin and its target proteinase, APR, and to compare these mutations with mutations at similar positions in the TIMPs. In particular, we were interested in Ser-2I, which is conserved among known serralysin inhibitors as well as in the TIMPs. This residue occupies the S1' pocket of the target proteinase (9, 13, 14), and its hydroxyl donates a hydrogen bond to the catalytic base, which is Glu-177P in APR. Our rationale for constructing the particular mutations in APRin position 2 is as follows.

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 ({Delta}{Delta}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 189–200), enabling formation of a {pi}-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.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of a University of Louisville Graduate Fellowship. Back

|| 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.

{ddagger}{ddagger} 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. Back

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. Back

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). Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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