Requirement for hydrophobic Phe residues in Pleurotus ostreatus proteinase A inhibitor 1 for stable inhibition

Shuichi Kojima,1 and Yuri Hisano

Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo 171-8588, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) has been shown to be unique among the various serine protease inhibitors in that its C-terminal region appears to be the reactive site responsible for its inhibitory action toward proteases. To investigate in more detail the mechanism of inhibition by POIA1, we have been studying its structural requirements for stable inhibition of proteases. In this study, we focused on hydrophobic Phe residues, which are generally located in the interior of protein molecules. A Phe->Ala replacement at position 44 or 56 was introduced into a `parent' mutant of POIA1 that had been converted into a strong and resistant inhibitor of subtilisin BPN' by replacement of its six C-terminal residues with those of the propeptide of subtilisin BPN' and the effects on inhibitory properties and structural stability were examined. Both of the mutated POIA1 molecules not only were found to exhibit decreased ability to bind to subtilisin BPN' (80-fold for the F44A mutant and 13-fold for the F56A mutant), but were also converted to temporary inhibitors that were degraded by the protease. The structural stability of the mutated POIA1 was also lowered, as shown by a 13°C decrease in melting temperature for the F56A mutant. In particular, the F44A mutant was found to lose its tertiary structure, as judged from the circular dichroism spectrum, demonstrating that Phe44 is a strict requirement for structural formation by the POIA1 molecule. These results clearly indicate that stabilization of POIA1 by hydrophobic residues in its molecular interior is required for stable inhibition of the protease. This requirement for a stable tertiary structure is shared with other serine protease inhibitors, but other structural requirements seem to differ, in that strong binding with the protease is required for POIA1 whereas conformational rigidity around the reactive site is essential for many other protease inhibitors.

Keywords: hydrophobic residue/Pleurotus ostreatus/protease inhibitor/protein stability/temporary inhibition


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Proteinaceous protease inhibitors are unique because they inhibit the proteolytic activity of proteases by the formation of a non-degraded stable complex in spite of possessing a substrate-like structure around the reactive site (Laskowski and Kato, 1980Go; Barrett and Salvesen, 1986Go). Research on the structural requirements in protease inhibitors for inhibitory activity may provide further information on their inhibitory mechanisms toward proteases (Coplen et al., 1990Go; Kojima et al., 1993Go; Jackson and Fersht, 1994Go), although the amino acid residues around the reactive site have been shown to determine the inhibitory specificity and strength of inhibitors (Courtney et al., 1985Go; Kojima et al., 1990Go; Longstaff et al., 1990Go; Grzesiak et al., 2000Go). Based on this, we have demonstrated by mutational analysis that conformational rigidity around the reactive site, conferred by a disulfide bond, and stability of the whole inhibitor molecule itself, maintained by interactions such as salt bridges and hydrophobic interactions, are needed for the stable inhibition of subtilisin BPN' by Streptomyces subtilisin inhibitor (SSI) (Tamura et al., 1991Go; Kojima et al., 1993Go, 1994Go). Similarly, several hydrophobic residues present in the molecular interior are required for bovine pancreatic trypsin inhibitor (BPTI) to function as a protease inhibitor (Coplen et al., 1990Go). These proteins are typical serine protease inhibitors that possess reactive sites in the central region of the molecule and exhibit standard inhibitory mechanisms (Laskowski and Kato, 1980Go).

In contrast, Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) (Dohmae et al., 1995Go) and yeast proteinase B inhibitor 2 (YIB2) (Maier et al., 1979Go) are unique in the serine protease inhibitor family, since their reactive sites appear to be located at their C-termini (Kojima et al., 1999Go, 2001Go). These proteins show sequence similarity (~30%) with the propeptides of subtilisins, which bind their cognate proteases by their C-termini (Bryan et al., 1995Go; Gallagher et al., 1995Go; Wang et al., 1995Go). We have demonstrated that the wild-type proteins of POIA1 and YIB2 are temporary inhibitors that are initially potent but are gradually degraded by the protease when subtilisin BPN' is used as the target protease; however, both can be converted to strong and resistant inhibitors of subtilisin by replacement of their C-terminal six residues with those of the propeptide (Kojima et al., 1999Go, 2001Go). Therefore, the inhibitory mechanisms of these proteins, which use their C-termini as the reactive site, may be different from those of other more typical serine protease inhibitors.

Based on these findings and considerations, we replaced the hydrophobic Phe residues of POIA1, which exist in different sites of the molecular interior, by less hydrophobic residues and examined the effects of the replacement on the inhibitory properties. It is generally thought that hydrophobic residues in the molecular interior of proteins are required for maintenance of their tertiary structure and thus that their replacement will result in a decrease in stability.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

Restriction enzymes and DNA-modifying enzymes were purchased from Takara Shuzo and Toyobo. Subtilisin BPN' was obtained from Sigma. Other chemicals were of reagent grade for biochemical research.

Site-directed mutagenesis

Codon replacement of Phe residues by Ala at position 44 or 56 in the POIA1 gene was carried out by oligonucleotide-directed mutagenesis (Kunkel, 1985Go). The EcoRI–HindIII fragment of the POIA1 gene, in which the C-terminal six residues of POIA1 had been replaced by those of the propeptide of subtilisin BPN', was inserted into the EcoRI–HindIII site of plasmid pTZ19U and single-stranded DNA containing uracil bases was prepared by infection of helper phage M13KO7 into Escherichia coli CJ236 transformed by the constructed plasmid. Mutations were confirmed by dideoxy sequencing of the mutated plasmid and the mutated POIA1 gene was inserted into the NcoI–BamHI site of pET11d.

Expression and purification of the mutated POIA1

Large-scale cultivation of E.coli BL21(DE3) transformed by the expression plasmid was carried out by essentially the same procedures as described previously (Kojima et al., 2001Go). The mutated POIA1 proteins expressed in the soluble fraction of E.coli were purified by three-step chromatography. The soluble fraction was subjected to ion-exchange chromatography (DE32) and the POIA1-containing fractions were applied to a Superdex 75pg column. To improve the purity in this study, the fractions that contained POIA1 were dialyzed against H2O, lyophilized and then subjected to a second ion-exchange chromatography on a Resource Q column (1 ml) with a 0–1 M NaCl gradient in 50 mM Tris–HCl (pH 8.0) on an FPLC system.

Elucidation of inhibitory properties of the mutated POIA1

Concentrations of subtilisin BPN' and mutated POIA1 were determined spectrophotometrically using an A280ISOdia1% of 11.7 (Matsubara et al., 1965Go) and a molar absorption coefficient of 2500 (Gill and von Hippel, 1989Go), respectively. The inhibitory activity of the mutated POIA1 toward subtilisin BPN' was measured using the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe p-nitroanilide and inhibitor constants were obtained from the inhibitory profiles, as described in previous papers (Kojima et al., 1999Go, 2001Go).

Electrophoretic analysis was carried out to investigate the molecular state of the mutated POIA1 in the mixture with the protease. Subtilisin BPN' at 364 nM and a 20-fold molar excess of Phe44->Ala mutant or a 5-fold molar excess of Phe56->Ala mutant were incubated at 25°C in 0.1 M Tris–HCl (pH 8.0). Subsequent procedures, including SDS–PAGE, were the same as described previously (Kojima et al., 1999Go, 2001Go).

Circular dichroism (CD) spectra measurements

CD spectra of POIA1 at 50 µM in 50 mM sodium phosphate buffer (pH 7.0) were measured with a JASCO-J720 CD spectrophotometer. The pathlength of the cell was 1 mm. The temperature was maintained by circulation of electrostatically controlled water through a jacket surrounding the cell.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design of mutation

As described in the Introduction, we attempted to examine the effects of replacement of the hydrophobic residues in the molecular interior of POIA1 on the inhibitory properties and thermal stability of the protein. The Phe residues at position 44 or 56 were selected as the amino acid residues to be replaced. We have demonstrated that replacement of Ala47 of the propeptide of subtilisin BPN' by Phe, which corresponds to position 44 of POIA1, resulted in partial formation of tertiary structure in the propeptide (Kojima et al., 1998Go) and therefore Phe44 of POIA1 was considered to be an important residue required for structure maintenance of POIA1. On the other hand, the tertiary structure of the propeptide of subtilisin BPN' in the complex indicates that Leu59, which corresponds to position 56 of POIA1, forms a hydrophobic core with several hydrophobic residues such as Val12, Ile30 and Leu51 (Gallagher et al., 1995Go); therefore, Phe56 of POIA1 was also thought to be required for the formation of a hydrophobic core in POIA1.

Since wild-type POIA1 is a temporary inhibitor when subtilisin BPN' is used as the target protease (Kojima et al., 2001Go), we thought that it would be difficult to evaluate the precise effects of these replacements on the properties of POIA1. Therefore, the Phe->Ala replacement at position 44 or 56 was introduced into a mutated POIA1 that had been converted to a stronger and more resistant inhibitor of subtilisin by replacement of its six C-terminal residues with those of the propeptide of subtilisin BPN' (Kojima et al., 2001Go). This C-terminal mutant of POIA1 is hereafter referred to as `parent POIA1' and the mutants in which the Phe->Ala replacement at position 44 or 56 was introduced were named simply the F44A or F56A mutants, respectively.

Inhibitory properties of the mutated POIA1

The inhibitory activity of the F44A or F56A mutants of POIA1 toward subtilisin BPN' was measured using a synthetic substrate at various incubation times after mixing mutated POIA1 with subtilisin. The results are shown in Figures 1Go and 2A and B, along with those for the parent POIA1. The parent POIA1, which shows strong inhibitory activity, exhibits little time-dependent decrease in inhibitory activity (Kojima et al., 2001Go), whereas both POIA1 mutants show not only weaker inhibitory activity toward subtilisin than the parent POIA1, but also their inhibitory activity decreases as the incubation time is increased.



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Fig. 1. Comparison of the inhibitory activities of the parent and mutated POIA1 at 0 min incubation. After subtilisin BPN' (364 nM) and various concentrations of POIA1 had been mixed in 0.1 M Tris–HCl (pH 8.0), a 100 µl aliquot was added immediately to a solution containing a synthetic substrate and the absorbance at 410 nm was monitored at 25°C. The inhibitory activity was obtained at the time when subtilisin–POIA1 complex formation had reached equilibrium. ({circ}) Parent POIA1; ({triangleup}) F44A mutant; ({square}) F56A mutant.

 
The inhibitor constants of the POIA1 mutants at incubation time zero were determined to be 2.2x10-9 and 3.6x10-10 M for the F44A and F56A mutants, respectively. Since the parent POIA1 was shown to possess an inhibitor constant of 2.8x10-11 M towards subtilisin BPN' (Kojima et al., 2001Go), the binding ability of POIA1 was weakened about 80- and 13-fold by the Phe->Ala replacement at positions 44 and 56, respectively, and the F44A mutant was a 7-fold weak inhibitor of subtilisin BPN' than the F56A mutant. In a similar way, Figure 2AGo and B indicate that the rate of decrease in inhibitory activity of the F44A mutant is faster than that of the F56A mutant, since the inhibitory activity of the F44A mutant is substantially lost after 30 min of incubation even at a molar ratio of 15, whereas the F56A mutant retains inhibitory activity at a molar ratio of 3 after 2 h of incubation.





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Fig. 2. Inhibitory activities of (A) F44A mutant and (B) F56A mutant and (C) their electrophoretic patterns in mixtures with subtilisin BPN'. At 0 ({circ}), 30 ({triangleup}), 60 ({square}) and 120 min ({triangledown}) of incubation at 25°C, a 100 µl aliquot was withdrawn from the mixture of subtilisin BPN' and mutated POIA1 described in Figure 1Go and inhibitory activity was measured at 25°C using a synthetic substrate. The data at 120 min for the F44A mutant and at 30 and 60 min for the F56A mutant are not shown for simplicity. For electrophoretic analysis, subtilisin BPN' at 364 nM and a 20-fold molar excess of the F44A mutant or a 5-fold molar excess of the F56A mutant were incubated at 25°C in 0.1 M Tris–HCl (pH 8.0). At the indicated times, the proteins in a 100 µl aliquot were precipitated with trichloroacetic acid and subjected to SDS–PAGE (acrylamide concentration 18.8%).

 
To investigate whether these time-dependent decreases in inhibitory activity of the mutants are a result of degradation by the protease, as in the case of temporary inhibitor mutants of other protease inhibitors such as SSI and YIB2 (Tamura et al., 1991Go; Kojima et al., 1993Go, 1994Go, 1999Go), mixtures of the POIA1 mutants with subtilisin were subjected to electrophoretic analysis. As shown in Figure 2CGo, the band of the F44A mutant of POIA1 disappeared after 30 min of incubation even at a molar ratio of 20, whereas the band of the F56A mutant at a molar ratio of 5 was still present after incubation for 120 min. These results clearly demonstrate that the F44A mutant is a more sensitive inhibitor for degradation than the F56A mutant and agree well with the results of the inhibitory activity measurements described above.

CD spectra of the mutated POIA1

To obtain information about the tertiary structure of the mutated POIA1 molecules, their CD spectra were measured. As shown in Figure 3AGo, the parent POIA1 exhibited a spectrum typical for a protein composed of both {alpha}-helices and ß-sheets. The F56A mutant was also found to show a similar spectrum to that of the parent POIA1, although its CD intensities were lower than those of the parent POIA1, suggesting that overall the tertiary structure of the F56A mutant is essentially retained. In contrast, the CD spectrum of the F44A demonstrates that this mutant exists in a random coil structure and that the tertiary structure of POIA1 was destroyed by the single amino acid replacement Phe44->Ala.




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Fig. 3. (A) CD spectra of the parent POIA1 (solid line), F44A mutant (dashed line) and F56A mutant (dot-dashed line) of POIA1 at 50 µM in 50 mM sodium phosphate (pH 7.0). (B) Temperature dependence of CD intensity at 220 nm, which is the intensity minimum in the spectra. ({circ}) Parent POIA1; ({triangleup}) F56A mutant.

 
Then, the temperature dependence of the CD intensity at 220 nm of the parent and the F56A mutant of POIA1 was investigated to compare their thermal stabilities. As shown in Figure 3BGo T,1/2, the temperature at which the midpoint of decreasing intensity occurs, of the parent POIA1 is 59°C, whereas that of the F56A mutant is 46°C, indicating a 13°C decrease in melting temperature.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
POIA1 is unique among various serine protease inhibitors in that it inhibits the activity of a cognate protease by binding its C-terminal region to the substrate-binding site of the protease (Kojima et al., 2001Go). Therefore, we considered that studies to investigate the structural requirements for strong and stable inhibition of the protease by POIA1 are worthwhile to clarify its inhibitory mechanisms and its differences from many other serine protease inhibitors (Laskowski and Kato, 1980Go; Barrett and Salvesen, 1986Go).

In this study, we focused on the hydrophobic Phe residues at positions 44 and 56, which are thought to exist in different hydrophobic environments. In contrast to the strong and stable inhibition of subtilisin BPN' by the parent POIA1, it was demonstrated that POIA1 was converted to a temporary inhibitor by replacement of the Phe residues at positions 44 or 56 by Ala. Among the two mutants, the F44A mutant is a weaker and more sensitive inhibitor for degradation than the F56A mutant. Furthermore, the stability of POIA1 was decreased by the replacements: a 13°C decrease in melting temperature was observed for the F56A mutant and the F44A mutant existed in a random coil structure. These results clearly indicate that Phe residues are required for the inhibitory action of POIA1 through stabilization of the tertiary structure of the inhibitor and that the contribution of Phe44 is larger than that of Phe56. The stabilization of the POIA1 molecule is closely related to its stable inhibition of the cognate protease.

For other serine protease inhibitors, such as SSI, BPTI and barley chymotrypsin inhibitor 2 (CI2), it has been shown that high stability of the inhibitor molecules is an important requirement for their inhibitory action (Coplen et al., 1990Go; Tamura et al., 1991Go; Jackson and Fersht, 1994Go; Kojima et al., 1994Go). When the specific interactions that stabilize the overall tertiary structure of these inhibitor molecules are destroyed by an amino acid replacement, the resultant mutant inhibitor was found to be converted to a temporary inhibitor that is degraded by the cognate protease, with lowering of its melting or denaturation temperature. Examples of such replacements include Trp86 in the molecular interior of SSI by His (Tamura et al., 1991Go), Arg29 of SSI (which forms a salt bridge with the C-terminus) by Ala (Kojima et al., 1994Go) and Phe22 or Phe33 in the hydrophobic core of BPTI by Ile (Coplen et al., 1990Go) (although additional removal of a disulfide bridge near the reactive site is needed for conversion to a temporary inhibitor in the case of BPTI). These findings clearly indicate that these inhibitor molecules are designed, by virtue of the many interactions in their molecules, to minimize the formation of denatured species that are susceptive to proteolysis. As a consequence, these molecules generally possess high stability.

Our results from this study show that the inhibitory mechanisms described above are also applicable to POIA1, although POIA1 is different from many other serine protease inhibitors in that it binds with the cognate protease by its C-terminal region. Therefore, we conclude that high molecular stability is a general requirement for the function of protease inhibitors.

In addition to high stability of the overall tertiary structure, conformational rigidity around the reactive site has also been suggested or demonstrated to be required for the inhibitory action of molecules such as SSI, CI2 and ovomucoid domain 3. It has been proposed or shown that a disulfide bridge (Kojima et al., 1993Go) or Asn-mediated hydrogen bonds in SSI (Takeuchi et al., 1991Go) or ovomucoid domain 3 (Ardelt and Laskowski, 1991Go) and hydrogen bonding and salt bridge network in CI2 (Jackson and Fersht, 1994Go) introduce conformational rigidity into their reactive sites and removal of these interactions by mutation results in their conversion to a temporary inhibitor.

In contrast, the C-terminal region of POIA1, which is the reactive site of the inhibitor, protrudes from the core structure of the POIA1 molecule and therefore is flexible in the isolated state if the tertiary structure of POIA1 resembles that of the propeptide of subtilisin BPN', which also binds with the protease by its C-terminal region (Bryan et al., 1995Go; Gallagher et al., 1995Go; Wang et al., 1995Go). Other unique points of the inhibitory mechanism of POIA1 are that this inhibitor lacks a region on the C-terminal side of the reactive site and that strong binding with the protease is required for resistance of the POIA1 molecule to proteolytic degradation. In fact, when the ability of POIA1 to bind to the protease was weakened by mutation, the resultant mutant was shown to be converted to a temporary inhibitor (Kojima et al., 2001Go). In contrast, it has been shown in the case of SSI that weak binding of a mutated inhibitor molecule with a protease does not necessarily lead to conversion to a temporary inhibitor, e.g. the Met73->Pro mutant at the P1 site of SSI (Kojima et al., 1991Go). These findings indicate that the differences in inhibitory mechanisms between POIA1 and many other serine protease inhibitors result in different requirements, in addition to stabilization of the overall tertiary structure of the inhibitor molecule, for stable inhibition of proteases: strong binding with the protease for POIA1 and conformational rigidity around the reactive site for many other inhibitors.

In the initial selection of mutations in this study, we considered that both Phe44 and Phe56 are important residues for the formation of the hydrophobic core. However, it was shown that the F44A mutant is more susceptive to proteolysis than the F56A mutant. In addition, the F44A mutant was found to lack tertiary structure, as judged from its CD spectrum. These results indicate that the contribution of Phe44 to the formation of the structure of POIA1 is much larger than that of Phe56. One plausible explanation for this is that the Ala47->Phe mutation in the propeptide of subtilisin BPN', which corresponds to Phe44 of POIA1, resulted in partial formation of tertiary structure of the propeptide in the isolated state. In contrast, the amino acid that corresponds to Phe56 of POIA1 is Leu in both YIB2 and the propeptide. This indicates that the presence of Phe at position 56 of POIA1 is not essential for structure formation and that Phe56 can be replaced by other hydrophobic residues from the structural viewpoints, although it is required for the stable inhibition of the protease.

Although the F44A mutant lacks the tertiary structure, it inhibited the protease to a meaningful extent (Ki = 2.2x10-9 M). This shows that the amino acid sequence of the C-terminal region of POIA1 is needed for initial binding of POIA1 with the protease and that the existence of tertiary structure in the core part of POIA1 is required for subsequent stable inhibition of the protease without degradation. As discussed above, the C-terminal region of POIA1 appears to be flexible even in the parent POIA1 possessing a defined tertiary structure and the conformation of the C-terminal region seems to be essentially the same in the F44A mutant, thus leading to expression of some inhibitory activity.


    Notes
 
1 To whom correspondence should be addressed. E-mail: shuichi.kojima{at}gakushuin.ac.jp Back


    Acknowledgments
 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and a Grant from the Novo Nordisk Research Foundation.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received December 5, 2001; accepted January 8, 2002.





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