The Staphostatin-staphopain complex: a forward binding inhibitor in complex with its target cysteine protease

Staphostatins are the endogenous inhibitors of the major secreted cysteine proteases of Staphylococcus aureus, the staphopains. Our recent crystal structure of staphostatin B has shown that this inhibitor forms a mixed, eight-stranded beta-barrel with statistically significant similarity to lipocalins, but not to cystatins. We now present the 1.8-A crystal structure of staphostatin B in complex with an inactive mutant of its target protease. The complex is held together through extensive interactions and buries a total surface area of 2300 A2. Unexpectedly for a cysteine protease inhibitor, staphostatin B binds to staphopain B in an almost substrate-like manner. The inhibitor polypeptide chain runs through the protease active site cleft in the forward direction, with residues IG-TS in P2 to P2' positions. Both in the free and complexed forms, the P1 glycine residue of the inhibitor is in a main chain conformation only accessible to glycines. Mutations in this residue lead to a loss of affinity of the inhibitor for protease and convert the inhibitor into a substrate.     

A comparison of staphostatin B with standard mechanism serine protease inhibitors

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.     

Crystal structure of wild-type human procathepsin K

Cathepsin K is a lysosomal cysteine protease belonging to the papain superfamily. It has been implicated as a major mediator of osteoclastic bone resorption. Wild-type human procathepsin K has been crystallized in a glycosylated and a deglycosylated form. The latter crystals diffract better, to 3.2 A resolution, and contain four molecules in the asymmetric unit. The structure was solved by molecular replacement and refined to an R-factor of 0.194. The N-terminal fragment of the proregion forms a globular domain while the C-terminal segment is extended and shows substantial flexibility. The proregion interacts with the enzyme along the substrate binding groove and along the proregion binding loop (residues Ser138-Asn156). It binds to the active site in the opposite direction to that of natural substrates. The overall binding mode of the proregion to cathepsin K is similar to that observed in cathepsin L, caricain, and cathepsin B, but there are local differences that likely contribute to the specificity of these proregions for their cognate enzymes. The main observed difference is in the position of the short helix alpha3p (67p-75p), which occupies the S' subsites. As in the other proenzymes, the proregion utilizes the S2 subsite for anchoring by placing a leucine side chain there, according to the specificity of cathepsin K toward its substrate.     

The staphostatin family of cysteine protease inhibitors in the genus Staphylococcus as an example of parallel evolution of protease and inhibitor specificity

Staphostatins constitute a family of staphylococcal cysteine protease inhibitors sharing a lipocalin-like fold and a unique mechanism of action. Each of these cytoplasmic proteins is co-expressed from one operon, together with a corresponding target extracellular cysteine protease (staphopain). To cast more light on staphostatin/staphopain interaction and the evolution of the encoding operons, we have cloned and characterized a staphopain (StpA2aur CH-91) and its inhibitor (StpinA2aur CH-91) from a novel staphylococcal thiol protease operon (stpAB2CH-91) identified in S. aureus strain CH-91. Furthermore, we have expressed a staphostatin from Staphylococcus warneri (StpinBwar) and characterized its target protease (StpBwar). Analysis of the reciprocal interactions among novel and previously described members of the staphostatin and staphopain families demonstrates that the co-transcribed protease is the primary target for each staphostatin. Nevertheless, the inhibitor derived from one species of Staphylococcus can inhibit the staphopain from another species, although the Ki values are generally higher and inhibition only occurs if both proteins belong to the same subgroup of either S. aureus staphopain A/staphostatin A (alpha group) or staphopain B/staphostatin B (beta group) orthologs. This indicates that both subgroups arose in a single event of ancestral allelic duplication, followed by parallel evolution of the protease/inhibitor pairs. The tight coevolution is likely the result of the known deleterious effects of uncontrolled staphopain action.     

Growth phase-dependent production of a cell wall-associated elastinolytic cysteine proteinase by Staphylococcus epidermidis

Staphylococcus epidermidis, a Gram-positive, coagulase-negative bacterium is a predominant inhabitant of human skin and mucous membranes. Recently, however, it has become one of the most important agents of hospital-acquired bacteriemia, as it has been found to be responsible for surgical wound infections developed in individuals with indwelling catheters or prosthetic devices, as well as in immunosupressed or neutropenic patients. Despite their medical significance, little is known about proteolytic enzymes of S. epidermidis and their possible contribution to the bacterium's pathogenicity; however, it is likely that they function as virulence factors in a manner similar to that proposed for the proteases of Staphylococcus aureus. Here we describe the purification of a cell wall-associated cysteine protease from S. epidermidis, its biochemical properties and specificity. A homology search using N-terminal sequence data revealed similarity to staphopain A (ScpA) and staphopain B (SspB), cysteine proteases from S. aureus. Moreover, the gene encoding S. epidermidis cysteine protease (Ecp) and a downstream gene coding for a putative inhibitor of the protease form an operon structure which resembles that of staphopain A in S. aureus. The active cysteine protease was detected on the bacterial cell surface as well as in the culture media and is apparently produced in a growth phase-dependent manner, with initial expression occurring in the mid-logarithmic phase. This enzyme, with elastinolytic properties, as well as the ability to cleave alpha1PI, fibrinogen and fibronectin, may possibly contribute to the invasiveness and pathogenic potential of S. epidermidis.     

Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU

On the basis of the structure of a HslUV complex, a mechanism of allosteric activation of the HslV protease, wherein binding of the HslU chaperone propagates a conformational change to the active site cleft of the protease, has been proposed. Here, the 3.1 A X-ray crystallographic structure of Haemophilus influenzae HslUV complexed with a vinyl sulfone inhibitor is described. The inhibitor, which reacts to form a covalent linkage to Thr1 of HslV, binds in an "antiparallel beta" manner, with hydrogen-bond interactions between the peptide backbone of the protease and that of the inhibitor, and with two leucinyl side chains of the inhibitor binding in the S1 and S3 specificity pockets of the protease. Comparison of the structure of the HslUV-inhibitor complex with that of HslV without inhibitor and in the absence of HslU reveals that backbone interactions would correctly position a substrate for cleavage in the HslUV complex, but not in the HslV protease alone, corroborating the proposed mechanism of allosteric activation. This activation mechanism differs from that of the eukaryotic proteasome, for which binding of activators opens a gated channel that controls access of substrates to the protease, but does not perturb the active site environment.     

Trypsin activity reduced by an autocatalytically produced nonapeptide

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native beta-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.     

Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and staphopain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains.     

Displacement of the occluding loop by the parasite protein, chagasin, results in efficient inhibition of human cathepsin B

Cathepsin B is a papain-like cysteine protease showing both endo- and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (Ki for H110A cathepsin B, 0.35 nm) due to an improved association rate (kon, 5 x 10(5) m(-1)s(-1)). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 angstroms resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.     

A thermostable cysteine protease precursor from a tropical plant contains an unusual C-terminal propeptide: cDNA cloning, sequence comparison and molecular modeling studies

We report here the cloning and characterization of the entire cDNA of a papain-like cysteine protease from a tropical flowering plant. The 1098-bp ORF of the cDNA codify a protease precursor having a signal peptide of 19 amino acids, a cathepsin-L like N-terminal proregion of 114 amino acids, a mature enzyme part of 208 amino acids and a C-terminal proregion of 24 amino acids. The derived amino acid sequence of the mature part tallies with the thermostable cysteine protease Ervatamin-C--as was aimed at. The C-terminal proregion of the protease has altogether a different sequence pattern not observed in other members of the family and it contains a negatively charged helical zone. The three-dimensional model of the precursor, based on the homology modeling and X-ray structure, shows that the extended peptide stretch region of the N-terminal propeptide, covering the interdomain cleft, contains protruding side chains of positively charged residues. This study also indicates that the negatively charged zone of C-terminal propeptide may interact with the positively charged zone of the N-terminal propeptide in a cooperative manner in the maturation process of this enzyme.     

Conformational lability in serine protease active sites: structures of hepatocyte growth factor activator (HGFA) alone and with the inhibitory domain from HGFA inhibitor-1B

Hepatocyte growth factor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. When activated HGF binds its cognate receptor Met, cellular signals lead to cell growth, differentiation, and migration, activities which promote tissue regeneration in liver, kidney and skin. Intervention in the conversion of HGF to its active form has the potential to provide therapeutic benefit where HGF/Met activity is associated with tumorigenesis. To help identify ways to moderate HGF/Met effects, we have determined the molecular structure of the protease domain of HGFA. The structure we determined, at 2.7 A resolution, with no pseudo-substrate or inhibitor bound is characterized by an unconventional conformation of key residues in the enzyme active site. In order to find whether this apparently non-enzymatically competent arrangement would persist in the presence of a strongly-interacting inhibitor, we also have determined, at 2.6 A resolution, the X-ray structure of HGFA complexed with the first Kunitz domain (KD1) from the physiological inhibitor hepatocyte growth factor activator inhibitor 1B (HAI-1B). In this complex we observe a rearranged substrate binding cleft that closely mirrors the cleft of other serine proteases, suggesting an extreme conformational dynamism. We also characterize the inhibition of 16 serine proteases by KD1, finding that the previously reported enzyme specificity of the intact extracellular region of HAI-1B resides in KD1 alone. We find that HGFA, matriptase, hepsin, plasma kallikrein and trypsin are potently inhibited, and use the complex structure to rationalize the structural basis of these results.     

Role of the proregion in the production and secretion of the Yarrowia lipolytica alkaline extracellular protease

The yeast Yarrowia lipolytica secretes an alkaline extracellular protease (AEP). It is first synthesized as a precursor comprising a putative signal peptide, a stretch of 10 X-Ala or X-Pro sequences that are substrates for a dipeptidyl aminopeptidase, a large pro-region that contains a glycosylation site and two Lys-Arg sites that can be cleaved by a KEX2-like endoprotease and finally the mature protease itself. A defect in the XPR6 (KEX2-like) gene results in the secretion of an inactive proenzyme (Matoba, S., and Ogrydziak, D. M. (1989) J. Biol. Chem. 264, 6037-6043), showing that the proregion inhibits protease activity. To determine whether the proregion plays an additional role in protease secretion, we have generated deletions and point mutations in the corresponding region of the structural gene. In this paper we examine the effects of these mutations on AEP secretion and maturation and show that the proregion is essential for its secretion. All deletions affecting the proregion resulted in the intracellular accumulation of unprocessed precursors. Deletion of the glycosylation site in the proregion resulted in the production of an unglycosylated precursor that was secreted and matured correctly at 18 degrees C but accumulated in the cells at 28 degrees C. From these results, we propose that the AEP prosequence plays an additional essential role in guiding the proper folding of the protein into a conformation compatible with secretion.     

Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine

Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site.     

Trypsin Activity Reduced by an Autocatalytically Produced Nonapeptide

Abstract

Trypsin, a serine protease enzyme plays a pivotal role in digestion and is autocatalytic. The crystal structure of a complex formed between porcine trypsin and an auto catalytically produced peptide is reported here. This complex shows a reduction in enzyme activity as compared to native β-trypsin. The nonapeptide has a lysine, which is recognized by Asp 189 at the specificity pocket. The auto catalytically produced native nonapeptide is bound at the active site cleft like other trypsin inhibitors but the important interactions with the oxyanion hole are absent. The peptide covers only a part of the active site cleft and hence the enzyme activity is reduced rather than being inhibited.     

Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition.

Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.     

Folding incompetence of cathepsin L-like cysteine proteases may be compensated by the highly conserved, domain-building N-terminal extension of the proregion

Folding of cathepsins L and S depend upon their proregion which extends the enzyme part by about 100 amino acids. Only a minority of the prosequence follows the structural template provided by the enzyme part; the majority forms an autonomous minidomain fairly distant from the active site cleft. We suggest that this prodomain may be the structural correlate of a foldase function of the proregion within the cathepsin L-like subfamily of papain-type cysteine proteases and report on a functional approach supporting this hypothesis.     

Structural comparison suggests that thermolysin and related neutral proteases undergo hinge-bending motion during catalysis.

Crystal structures are known for three members of the bacterial neutral protease family: thermolysin from Bacillus thermoproteolyticus (TLN), the neutral protease from Bacillus cereus (NEU), and the elastase of Pseudomonas aeruginosa (PAE), both in free and ligand-bound forms. Each enzyme consists of an N-terminal and C-terminal domain with the active site formed at the junction of the two domains. Comparison of the different molecules reveals that the structure within each domain is well conserved, but there are substantial hinge-bending displacements (up to 16 degrees) of one domain relative to the other. These domain motions can be correlated with the presence or absence of bound inhibitor, as was previously observed in the specific example of PAE [Thayer, M.M., Flaherty, K.M., & McKay, D.B. (1991) J. Biol. Chem. 266, 2864-2871]. The binding of inhibitor appears to be associated with a reduction of the domain hinge-bending angle by 6-14 degrees and a closure of the "jaws" of the active site cleft by about 2 A. Crystallographic refinement of the structure of thermolysin suggests that electron density seen in the active site of the enzyme in the original structure determination probably corresponds to a bound dipeptide. Thus, the crystal structure appears to correspond to an enzyme-inhibitor or enzyme-product complex, rather than the free enzyme, as has previously been assumed.     

Mechanism of Aurora B activation by INCENP and inhibition by hesperadin

Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.     

Crystal structure of thrombin-ecotin reveals conformational changes and extended interactions

The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases. A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin. The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding. Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions. The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters). The preference for a Val at P4 is clearly defined. The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites.     

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), a modified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 A crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent approximately 15 degrees to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.