A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

Complex of subtilisin BPN' with Streptomyces subtilisin inhibitor. Complex formation concomitant with change in reducibility of disulfide bonds in the inhibitor

Structure of the complex of Streptomyces subtilisin inhibitor (SSI) with subtilisin BPN' was studied by examining the thermal denaturation and reducibility of disulfide bonds. The denaturation temperature of the complex was significantly higher than that of the enzyme. Two disulfide bonds localized in the inhibitor side were completely reduced in the complex, whereas only one of them was reduced in the free SSI. Gel filtration of the reduced complex solution showed clearly that the main products of reduction of the complex were two peptide fragments of SSI divided at the active site. The resistive disulfide bond in the complexed inhibitor became accessible as a result of a large conformational change due to splitting of the half-reduced inhibitor.     

Refined crystal structure of the complex of subtilisin BPN' and Streptomyces subtilisin inhibitor at 1.8 A resolution

The crystal structure of subtilisin BPN' complexed with a proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor) was refined at 1.8 A resolution to an R-factor of 0.177 with a root-mean-square deviation from ideal bond lengths of 0.014 A. The work finally established that the SSI-subtilisin complex is a Michaelis complex with a distance between the O gamma of active Ser221 and the carbonyl carbon of the scissile peptide bond being an intermediate value between a covalent bond and a van der Waals' contact, 2.7 A. This feature, as well as the geometry of the catalytic triad and the oxyanion hole, is coincident with that found in other highly refined crystal structures of the complex of subtilisin Novo, subtilisin Carlsberg, bovine trypsin or Streptomyces griseus protease B with their proteinaceous inhibitors. The enzyme-inhibitor beta-sheet interaction is composed of two separate parts: that between the P1-P3 residues of SSI and the 125-127 chain segment (the "S1-3 site") of subtilisin and that between the P4-P6 residues of SSI and th 102-104 chain segment (the "S4-6 site") of subtilisin. The latter beta-interaction is unique to subtilisin. In contrast, the beta-sheet interaction previously found in the complex of subtilisin Novo and chymotrypsin inhibitor 2 or in the complex of subtilisin Carlsberg and Eglin C is distinct from the present complex in that the two types of beta-interactions are not separate. As for the flexibility of the molecules comprising the present complex, the following observations were made by comparing the B-factors for free and complexed SSI and comparing those for free and complexed subtilisin BPN'. The rigidification of the component molecules upon complex formation occurs in a very localized region: in SSI, the "primary" and "secondary" contact regions and the flanking region; in subtilisin BPN', the S1-3 and S4-6 sites and the flanking region.     

Crystal structure of the complex of subtilisin BPN' with its protein inhibitor Streptomyces subtilisin inhibitor. The structure at 4.3 Angstroms resolution

The crystal structure of the complex of subtilisin BPN′ (EC 3.4.21.14) with its protein inhibitor (Streptomyces subtilisin inhibitor) was solved at 4.3 Å resolution, thus establishing the following. (1) Two subtilisin BPN′ molecules (2E) associate with one dimeric inhibitor molecule (I₂) to form the complex molecule E₂I₂. (2) The conformation of neither the inhibitor nor subtilisin BPN′ undergoes any detectable change at this resolution upon complex formation. (3) The inhibitor binds to subtilisin to form an antiparallel β-sheet, as in the case of trypsin/ trypsin inhibitor complexes. (4) The scissible bond of the inhibitor is between Met73′ and Val74′, as proposed earlier (Ikenaka et al., 1974). (5) The protein inhibitor and the substrates bind to subtilisin BPN′ in essentially the same way.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

Characterization of recombinant C1 inhibitor P1 variants.

Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties. The ability to bind to target proteases, as well as potential substrate-like behavior, was investigated with radioimmunoassays. The P1-Lys variant retained binding capacity toward C1s, plasmin, and kallikrein. In addition, complex formation with C1s was detected for P1-Asn and P1-His. All other P1 substitutions resulted in C1 inhibitor variants that neither complexed with nor were inactivated by C1s, kallikrein, beta-factor XIIa, or plasmin. Electrophoretic studies confirmed that P1-Lys and P1-His can form sodium dodecyl sulfate-resistant complexes with C1s. In contrast, the C1s-P1-Asn complex dissociated upon addition of sodium dodecyl sulfate. Kinetic experiments by the method of progress curves generated association rate constants (kon) with C1s of 4.2 x 10(4) M-1 s-1 for recombinant wild-type C1 inhibitor and 1.7 x 10(4) M-1 s-1 for P1-Lys. For P1-Asn and P1-His, kon was decreased approximately 100-fold. The results from inhibition experiments were compatible with a model of reversible inhibition, although the observed dissociation rate for wild-type C1 inhibitor is too low (1-2 x 10(-6) s-1) to be physiologically relevant. The overall inhibition constant (Ki) was estimated to be 0.03 nM. With P1-Asn, reversible inhibition could be demonstrated directly upon dilution of preformed complexes; the observed dissociation rate constant was 3.2 x 10(-4) s-1; and Ki increased to approximately 380 nM. These findings are discussed in relation to inhibitor specificity and inhibition mechanism.     

Chemical replacement of P1' serine residue at the second reactive site of soybean protease inhibitor C-II

The P1' Ser(50) at the second reactive site of soybean protease inhibitor C-II was replaced with arginine to confirm the contribution of this residue to the inhibition. The Arg derivative had less trypsin inhibitory activity (Ki = 1 X 10(-7) M) than the Ser derivative (Ki = 2 X 10(-8) M), in contrast to the results obtained from studies on peanut protease inhibitor B-III reported in the previous paper (J. Biochem. 101, 723-728 (1987)). These results suggest that each Bowman-Birk type inhibitor has an amino acid at the P1' position inherently best suited to maintaining its inhibitory activity, and that serine is not unique for the P1' amino acid in Bowman-Birk type inhibitors.     

Chemical replacement of P1' arginine residue at the first reactive site of peanut protease inhibitor B-III

The contribution of the P1' residue at the first reactive site of peanut protease inhibitor B-III to the inhibition was analyzed by replacement of the P1' Arg(11) with other amino acids (Arg, Ser, Ala, Leu, Phe, Asp) after selective modification of the second reactive site. The Arg derivative had the same trypsin inhibitory activity as the native inhibitor (Ki = 2 X 10(-9) M). The Ser derivative inhibited more weakly (Ki = 2 X 10(-8) M). The Ala and Leu derivatives inhibited trypsin very weakly (Ki = 2 X 10(-7) M and 4 X 10(-7) M, respectively), and the Phe and Asp derivatives not at all. These results suggest that the P1' arginine residue is best for inhibitory activity at the first reactive site of B-III, although it has been suggested that a P1' serine residue at the reactive site is best for inhibitory activity of Bowman-Birk type inhibitors.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

Interaction of trypsin-like protease from Streptomyces griseus with an immobilized inhibitor from kidney bean.

An immobilized double-headed inhibitor from Phaseolus vulgaris L. selectively binds the trypsin-like enzyme produced by Streptomyces griseus. Binding takes place at pH 8.0, and at pH 2.0 the protease can be quantitatively released from the complex. Purified by affinity chromatography, the trypsin-like enzyme is homogeneous according to polyacrylamide gel electrophoresis and ultracentrifugation data. Physico-chemical and enzymic properties of the enzyme are identical to those exhibited by the enzyme purified by ion-exchange chromatography. Chymoelastases from Str. griseus as well as the subtilisin-like enzyme do not interact with an immobilized inhibitor. In solution, the inhibitor from P. vulgaris gives a stable ternary complex with bovine trypsin and chymotrypsin, whereas with an immobilized inhibitor the trypsin, if present, tends to displace chymotrypsin in an chymotrypsin inhibitor complex. This evidence suggests that immobilization results in considerable changes in inhibitor properties.     

Effect of inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficiently inhibits a bacterial serine protease, subtilisin BPN'. We recently demonstrated that functional change in SSI was possible simply by replacing the amino acid residue at the reactive P1 site (methionine 73) of SSI. The present paper reports the additional effect of replacing methionine 70 at the P4 site of SSI (Lys73) on inhibitory activity toward two types of serine proteases, trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversion of methionine 70 at the P4 site of SSI(Lys73) to glycine or alanine resulted in increased inhibitory activity toward trypsin and lysyl endopeptidase, while replacement with phenylalanine weakened the inhibitory activity toward trypsin. This suggests that steric hindrance at the P4 site of SSI(Lys73) is an obstacle for its binding with trypsin. In contrast, the same P4 replacements had hardly any effect on inhibitory activity toward subtilisin BPN'. Thus the subsite structure of subtilisin BPN' is tolerant to these replacements. This contrast in the effect of P4 substitution might be due to the differences in the S4 subsite structures between the trypsin-like and the subtilisin-like proteases. These findings demonstrate the importance of considering structural complementarity, not only at the main reactive site but also at subsites of a protease, when designing stronger inhibitors.     

Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.     

The coupling of catalytically relevant conformational fluctuations in subtilisin BPN' to solution viscosity revealed by hydrogen isotope exchange and inhibitor binding

We have measured the tritium outexchange of subtilisin BPN'. A consistent and rather small group of hydrogens was isolated by their sensitivity to inhibitor binding. The viscosity dependence of exchange from these inhibitor protected hydrogens was then examined in 0.05 M MES buffer, pH 6.5 and 10 degrees C. The viscosity of the reaction medium was varied by added glycerol and ethylene glycol. The exchange rates were corrected to be compared at identical hydroxyl ion and water activity. The salient observation is the strikingly similar viscosity coupling behavior when compared to the deacylation step of ester hydrolysis catalyzed by the same enzyme (Ng and Rosenberg, Biophysical Chemistry, 39 (1991) 57). We have obtained a viscosity coupling constant of 0.68 -/+ 0.18 for hydrogen exchange in glycerol (cf. 0.65 -/+ 0.11 for deacylation in glycerol, sucrose, glucose and fructose); 1.67 -/+ 0.07 for outexchange (cf. 1.92 -/+ 0.09 for deacylation), in the presence of ethylene glycol. The two reactions are very chemically dissimilar, yet they show very similar viscosity coupling behavior. This together with the well established role of structural fluctuations in hydrogen exchange implies a similar role of structural fluctuations in the deacylation step of subtilisin BPN' catalyzed ester hydrolysis.     

Protective properties of recombinant IgA1 protease from meningococcus

The study of enzymatic and protective properties of recombinant IgA1 protease in active and mutant form has shown that the active form of IgA1 protease exhibited species- and type-specificity for mouse and human immunoglobulins. A mutant form, lacking enzymatic activity, had protective properties against meningococcal infection, induced by meningococcus serogroup A, B and C; it protected mice from lethal infection by live virulent cultures of heterologous serogroups of meningococcus. The results obtained in this study suggest that IgA1 protease may be considered as a perspective preparation at the stages of development of a polyvalent vaccine for protection of human against meningococcal infections of various etiology.     

Interaction of F1-ATPase, from ox heart mitochondria with its naturally occurring inhibitor protein. Studies using radio-iodinated inhibitor protein

The ox heart mitochondrial inhibitor protein may be iodinated with up to 0.8 mol 125I per mol inhibitor with no loss of inhibitory activity, with no change in binding affinity to submitochondrial particles, and without alteration in the response of membrane-bound inhibitor to energisation. Tryptic peptide maps reveal a single labelled peptide, consistent with modification of the single tyrosine residue of the protein. A single type of high-affinity binding site (Kd=96 . 10 (-9)M) for the inhibitor protein has been measured in submitochondrial particles. The concentration of this site is proportional to the amount of membrane-bound F1, and there appears to be one such site per F1 molecule. The ATp hydrolytic activity of submitochondrial particles is inversely proportional to the occupancy of the high-affinity binding site for the inhibitor protein. No evidence is found for a non-inhibitory binding site on the membrane or on other mitochondrial proteins. In intact mitochondria from bovine heart, the inhibitor protein is present in an approx. 1:1 ratio with F1. Submitochondrial particles prepared by sonication of these mitochondria with MgATP contain about 0.75 mol inhibitor protein per mol F1, and show about 25% of the ATPase activity of inhibitor-free submitochondrial particles. Additional inhibitor protein can be bound to these particles to a level of 0.2 mol/mol F1, with consequent loss of ATPase activity. If MgATP is omitted from the medium, or inhibitors of ATP hydrolysis are present, the rate of combination between F1 and its inhibitor protein is very much reduced. The equilibrium level of binding is, however, unaltered. These results suggest the presence of a single, high-affinity, inhibitory binding site for inhibitor protein on membrane-bound F1. The energisation of coupled submitochondrial particles by succinate oxidation or by ATP hydrolysis results in both the dissociation of inhibitor protein into solution, and the activation of ATP hydrolysis. At least 80% of the membrane-bound F1-inhibitor complex responds to this energisation by participating in a new equilibrium between bound and free inhibitor protein. This finding suggests that a delocalised energy pool is important in promoting inhibitor protein release from F1. Dissipation of the electrochemical gradient by uncouplers, or the binding of oligomycin or efrapetin effectively blocks energised release of the inhibitor protein. Conversely, the addition of aurovertin or adenosine 5'--[beta, lambda--imido]triphosphate enhances energy-driven release. The mode of action of various inhibitors on binding and energised release of the protein inhibitor is discussed.     

Overexpression in Escherichia coli and purification of recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm

Present research provided an efficient approach to obtain large quantities of active recombinant CI-b1, a Kunitz-type chymotrypsin inhibitor of silkworm, Bombyx mori. The cDNA encoding mature CI-b1 was cloned into pDEST17 vector. Recombinant protein with hexa-histidine tag attached to the N-terminal of CI-b1 was expressed in Escherichia coli Origami B cells. It can be purified to homogeneity via the gel filtration chromatography on a Sephacryl S-200 column followed the affinity chromatography on a Ni-NTA column. The two sequential purification procedures yielded 4.3mg purified (His)(6)-tagged CI-b1 from 200ml of culture medium. Studies on (His)(6)-tagged CI-b1 revealed that three disulfide bonds were formed in the recombinant CI-b1 and the inhibitory properties of recombinant CI-b1 against alpha-chymotrypsin were similar to those of native CI-b1. Recombinant CI-b1 immobilized on Ni-NTA resin was used to detect the interactions occurring between the CI-b1 and its target factors.