The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.     

N-terminal extension of the yeast IA3 aspartic proteinase inhibitor relaxes the strict intrinsic selectivity

Yeast IA(3) aspartic proteinase inhibitor operates through an unprecedented mechanism and exhibits a remarkable specificity for one target enzyme, saccharopepsin. Even aspartic proteinases that are very closely similar to saccharopepsin (e.g. the vacuolar enzyme from Pichia pastoris) are not susceptible to significant inhibition. The Pichia proteinase was selected as the target for initial attempts to engineer IA(3) to re-design the specificity. The IA(3) polypeptides from Saccharomyces cerevisiae and Saccharomyces castellii differ considerably in sequence. Alterations made by deletion or exchange of the residues in the C-terminal segment of these polypeptides had only minor effects. By contrast, extension of each of these wild-type and chimaeric polypeptides at its N-terminus by an MK(H)(7)MQ sequence generated inhibitors that displayed subnanomolar potency towards the Pichia enzyme. This gain-in-function was completely reversed upon removal of the extension sequence by exopeptidase trimming. Capture of the potentially positively charged aromatic histidine residues of the extension by remote, negatively charged side-chains, which were identified in the Pichia enzyme by modelling, may increase the local IA(3) concentration and create an anchor that enables the N-terminal segment residues to be harboured in closer proximity to the enzyme active site, thus promoting their interaction. In saccharopepsin, some of the counterpart residues are different and, consistent with this, the N-terminal extension of each IA(3) polypeptide was without major effect on the potency of interaction with saccharopepsin. In this way, it is possible to convert IA(3) polypeptides that display little affinity for the Pichia enzyme into potent inhibitors of this proteinase and thus broaden the target selectivity of this remarkable small protein.     

IA3, an aspartic proteinase inhibitor from Saccharomyces cerevisiae, is intrinsically unstructured in solution

IA(3) is a highly specific and potent 68-amino acid endogenous inhibitor of yeast proteinase A (YprA), and X-ray crystallographic studies have shown that IA(3) binds to YprA as an alpha-helix [Li, M., Phylip, L. H., Lees, W. E., Winther, J. R., Dunn, B. M., Wlodawer, A., Kay, J., and Gustchina, A. (2000) Nat. Struct. Biol. 7, 113-117]. Surprisingly, only residues 2-32 of IA(3) are seen in the X-ray structure, and the remaining residues are believed to be disordered in the complex. We have used circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy to show that IA(3) is unstructured in the absence of YprA. Specifically, IA(3) produced a CD spectrum characteristic of an unstructured peptide, and the (15)N HSQC NMR spectra of IA(3) were characteristic of a polypeptide lacking intrinsic structure. We characterized the unstructured state of IA(3) by using singular-value decomposition (SVD) to analyze the CD data in the presence of TFE, by fully assigning the unbound IA(3) protein by NMR and comparing the chemical shifts to published random-coil values, and by measuring (1)H-(15)N heteronuclear NOEs, which are all consistent with an unfolded protein. The IA(3) samples used for NMR analyses were active and inhibited YprA with an inhibition constant (K(i)) of 1.7 nM, and the addition of YprA led to a large spectral transition in IA(3). Calorimetric (ITC) data also show that the overall enthalpy of the interaction between IA(3) and YprA is exothermic.     

Inhibition of the aspartic proteinase from HIV-2

Kinetic constants were determined for the interaction of the HIV-2 aspartic proteinase with a synthetic substrate and a number of inhibitors at several pH values. Acetyl-pepstatin was more effective towards HIV-2 proteinase than the renin inhibitor, H-261; this effect is exactly the opposite from that observed previously for the proteinase from the HIV-1 AIDS virus.     

Stereochemical analysis of peptide bond hydrolysis catalyzed by the aspartic proteinase penicillopepsin

The X-ray crystal structures of native penicillopepsin and of its complex with a synthetic analogue of the inhibitor pepstatin have been refined recently at 1.8-A resolution. These highly refined structures permit a detailed examination of peptide hydrolysis in the aspartic proteinases. Complexes of penicillopepsin with substrate and catalytic intermediates were modeled, by using computer graphics, with minimal perturbation of the observed inhibitor complex. A thallium ion binding experiment shows that the position of solvent molecule O39, between Asp-33(32) and Asp-213(215) in the native structure, is favorable for cations, a fact that places constraints on possible mechanisms. A mechanism for hydrolysis is proposed in which Asp-213(215) acts as an electrophile by protonating the carbonyl oxygen of the substrate, thereby polarizing the carbon-oxygen bond, a water molecule bound to Asp-33(32) (O284 in the native structure) attacks the carbonyl carbon as the nucleophile in a general-base mechanism, the newly pyramidal peptide nitrogen is protonated, either from the solvent after nitrogen inversion or by an internal proton transfer via Asp-213(215) from a hydroxyl of the tetrahedral carbon, and the tetrahedral intermediate breaks down in a manner consistent with the stereoelectronic hypothesis. The models permit the rationalization of observed subsite preferences for substrates and may be useful in predicting subsite preferences of other aspartic proteinases.     

cDNA cloning of an aspartic proteinase secreted by Candida albicans.

cDNA of an aspartic proteinase secreted by Candida albicans No. 114 was isolated using the polymerase chain reaction (PCR). The primary structure of the enzyme was deduced from the nucleotide sequence of the cDNA and compared with the structures of Saccharomyces cerevisiae proteinase A and vacuolar aspartyl proteinase of C. albicans. The mature aspartic proteinase consisted of 341 amino acid residues, and was 17.6 and 15.3% identical with the proteinase A and the aspartyl proteinase, respectively. Two active aspartic acid sites and the amino acids near those sites were conserved in the aspartic proteinase. We also showed that there is another gene of aspartic proteinase than that of strain ATCC10231 reported by Hube et al (J. Med. Vet. Mycol. 29 (1991)) in the same C. albicans genome, both in that strain and in No. 114.     

Cleavage of human big endothelin-1 by Candida albicans aspartic proteinase

Abstract A Candida albicans aspartic proteinase (CAP), one of the secretory proteinases of Candida albicans , is thought to be a possible virulence factor in Candida albicans infection. Whereas endothelin-1 is found as an endothelium-derived strong vasoconstrictive peptide, it is known to have a role in the maintenance of vascular homeostasis and tissue survival. Endothelin-1 is generated from a precursor form of endothelin-1, the so-called big endothelin-1. It has recently been reported that cathepsin D, E and pepsin, which are aspartic proteinases, convert big endothelin-1 to endothelin-1. In this study, the relationship between CAP and big endothelin-1 was studied. High performance liquid chromatography analysis revealed that big endothelin-1 was cleaved into several amino acid sites by CAP, but endothelin-1 was not converted from big endothelin-1. CAP cleaved big endothelin-1 at different sites when compared with that of other known aspartic proteinases, and it suppressed endothelin-1 production through the degradation of big endothelin-1. CAP may break homeostatic mechanism of endothelin-1 in Candida albicans infectious lesion.     

The substrate specificity of SARS coronavirus 3C-like proteinase

The 3C-like proteinase of severe acute respiratory syndrome coronavirus (SARS) has been proposed to be a key target for structural based drug design against SARS. We have designed and synthesized 34 peptide substrates and determined their hydrolysis activities. The conserved core sequence of the native cleavage site is optimized for high hydrolysis activity. Residues at position P4, P3, and P3' are critical for substrate recognition and binding, and increment of beta-sheet conformation tendency is also helpful. A comparative molecular field analysis (CoMFA) model was constructed. Based on the mutation data and CoMFA model, a multiply mutated octapeptide S24 was designed for higher activity. The experimentally determined hydrolysis activity of S24 is the highest in all designed substrates and is close to that predicted by CoMFA. These results offer helpful information for the research on the mechanism of substrate recognition of coronavirus 3C-like proteinase.     

The proteinase yscA-inhibitor, IA3, gene. Studies of cytoplasmic proteinase inhibitor deficiency on yeast physiology

The gene of the proteinase yscA inhibitor IA3, PAI3, of the yeast Saccharomyces cerevisiae was isolated by oligonucleotide screening of a genomic DNA library and sequenced. The gene codes for a single protein of 68 amino acids. The structural PAI3 gene was deleted in vitro by oligonucleotide-site-directed mutagenesis. The mutated allele was introduced via homologous recombination into the genome of wild-type yeast and into the genome of a yeast mutant, which lacks the second cytoplasmic proteinase-inhibitor, IB2. The deficiency of either or of both inhibitors has no effect on the cell viability under various physiological conditions. The inhibitor mutants, however, show an increase in the general in vivo protein degradation rate. The IA3 mutant has a 2-3-fold increased protein degradation rate in the first 6 h after a shift from rich medium onto starvation-medium, whereas the IB2 mutant shows a constantly increased degradation rate of 20-50% under the same conditions. The inhibitor double null mutant has the same protein degradation rate as the IA3 null mutant. These results suggest an in vivo interaction between the vacuolor endopeptidases and their cytoplasmic inhibitors.     

Activity and inhibition of plasmepsin IV, a new aspartic proteinase from the malaria parasite, Plasmodium falciparum

A new aspartic proteinase from the human malaria parasite Plasmodium falciparum is able to hydrolyse human haemoglobin at a site known to be the essential primary cleavage site in the haemoglobin degradation pathway. Thus, plasmepsin IV may play a crucial role in this critical process which yields nutrients for parasite growth. Furthermore, synthetic inhibitors known to inhibit parasite growth in red cells in culture are able to inhibit the activity of this enzyme in vitro. As a result, plasmepsin IV appears to be a potential target for the development of new antiparasitic drugs.     

Switching the substrate specificity of the two-component NS2B-NS3 flavivirus proteinase by structure-based mutagenesis

The flavivirus NS2B-NS3(pro)teinase is an essential element in the proteolytic processing of the viral precursor polyprotein and therefore a potential drug target. Recently, crystal structures and substrate preferences of NS2B-NS3pro from Dengue and West Nile viruses (DV and WNV) were determined. We established that the presence of Gly-Gly at the P1'-P2' positions is optimal for cleavage by WNV NS3pro, whereas DV NS3pro tolerates well the presence of bulky residues at either P1' or P2'. Structure-based modeling suggests that Arg(76) and Pro(131)-Thr(132) limit the P1'-P2' subsites and restrict the cleavage preferences of the WNV enzyme. In turn, Leu(76) and Lys(131)-Pro(132) widen the specificity of DV NS3pro. Guided by these structural models, we expressed and purified mutant WNV NS2B-NS3pro and evaluated cleavage preferences by using positional scanning of the substrate peptides in which the P4-P1 and the P3'-P4' positions were fixed and the P1' and P2' positions were each randomized. We established that WNV R76L and P131K-T132P mutants acquired DV-like cleavage preferences, whereas T52V had no significant effect. Our work is the first instance of engineering a viral proteinase with switched cleavage preferences and should provide valuable data for the design of optimized substrates and substrate-based selective inhibitors of flaviviral proteinases.     

Structural features determining the site specificity of a rat liver cAMP-independent protein kinase.

The Seryl and Threonyl residues affected in α_s1 and in β-caseins by rat liver “casein kinase TS” (a cytosolic cAMP-independent protein kinase) have been identified. All of them, as well as the residues affected by the same enzyme in α_s2-casein are characterized by an acidic group two residues to their C terminus and by being located within predicted β-turns. Several other potential sites of phosphorylation, according to their primary structure, but located outside predicted β-turns, are not significantly labeled by the protein kinase. It seems conceivable therefore that both a definite aminoacid sequence including a critical acidic residue, and the existence of a β-turn are required for the activity of this protein kinase.     

Processing, activity, and inhibition of recombinant cyprosin, an aspartic proteinase from cardoon (Cynara cardunculus).

The cDNA encoding the precursor of an aspartic proteinase from the flowers of the cardoon, Cynara cardunculus, was expressed in Pichia pastoris, and the recombinant, mature cyprosin that accumulated in the culture medium was purified and characterized. The resultant mixture of microheterogeneous forms was shown to consist of glycosylated heavy chains (34 or 32 kDa) plus associated light chains with molecular weights in the region of 14,000-18,000, resulting from excision of most, but not all, of the 104 residues contributed by the unique region known as the plant specific insert. SDS-polyacrylamide gel electrophoresis under non-reducing conditions indicated that disulfide bonding held the heavy and light chains together in the heterodimeric enzyme forms. In contrast, when a construct was expressed in which the nucleotides encoding the 104 residues of the plant specific insert were deleted, the inactive, unprocessed precursor form (procyprosin) accumulated, indicating that the plant-specific insert has a role in ensuring that the nascent polypeptide is folded properly and rendered capable of being activated to generate mature, active proteinase. Kinetic parameters were derived for the hydrolysis of a synthetic peptide substrate by wild-type, recombinant cyprosin at a variety of pH and temperature values and the subsite requirements of the enzyme were mapped using a systematic series of synthetic inhibitors. The significance is discussed of the susceptibility of cyprosin to inhibitors of human immunodeficiency virus proteinase and particularly of renin, some of which were found to have subnanomolar potencies against the plant enzyme.     

Identification of a new aspartic proteinase expressed by the outer chorionic cell layer of the equine placenta

The pregnancy-associated glycoproteins (PAGs) are placental antigens that were initially characterized as pregnancy markers in the maternal circulation of domestic ruminant species. They are members of the aspartic proteinase gene family, having greatest sequence identity with pepsinogens. However, some are not capable of functioning as enzymes. The PAGs are associated with a large gene family within the Artiodactyla order (cattle, camels, pigs). So far, no members of this family have been characterized in species outside this order. This report describes the cloning and initial characterization of a PAG-like protein (equine PAG or ePAG) expressed in the placenta of the horse and zebra (order Perrisodactyla). Equine PAG is a proteinase capable of degrading 14C-hemoglobin and catalyzing the removal of its own pro-peptide. The ePAG mRNA is restricted to the chorion both prior to implantation and in the term placenta. Equine PAG is secreted from cultured placental tissue as both a processed (mature) and unprocessed (zymogen) form. Equine PAG shares similar identity with the PAGs and pepsinogens and probably arose from a pepsinogen-like precursor that gained the ability to be expressed in the placenta. The promoter of the ePAG gene shares sequence identity with the promoter from a bovine PAG gene but not with promoters of other aspartic proteinases. Therefore, we hypothesize that ePAG is a remnant of the pepsinogen-like progenitor gene that was expanded within the Artiodactyla to create the large and highly diverse PAG family.     

Processing of a cellular polypeptide by 3CD proteinase is required for poliovirus ribonucleoprotein complex formation.

Poliovirus interactions with host cells were investigated by studying the formation of ribonucleoprotein complexes at the 3' end of poliovirus negative-strand RNA which are presumed to be involved in viral RNA synthesis. It was previously shown that two host cell proteins with molecular masses of 36 and 38 kDa bind to the 3' end of viral negative-strand RNA at approximately 3 to 4 h after infection. We tested the hypothesis that preexisting cellular proteins are modified during the course of infection and are subsequently recruited to play a role in viral replication. It was demonstrated that the 38-kDa protein, either directly or indirectly, is the product of processing by poliovirus 3CD/3C proteinase. Only the modified 38-kDa protein, not its precursor protein, has a high affinity for binding to the 3' end of viral negative-strand RNA. This modification depends on proteolytically active proteinase, and a direct correlation between the levels of 3CD proteinase and the 38-kDa protein was demonstrated in infected tissue culture cells. The nucleotide (nt) 5-10 region (positive-strand numbers) of poliovirus negative-strand RNA is important for binding of the 38-kDa protein. Deletion of the nt 5-10 region in full-length, positive-strand RNA renders the RNA noninfectious in transfection experiments. These results suggest that poliovirus 3CD/3C proteinase processes a cellular protein which then plays an essential role during the viral life cycle.     

A heme-binding aspartic proteinase from the eggs of the hard tick Boophilus microplus

An aspartic proteinase that binds heme with a 1:1 stoichiometry was isolated and cloned from the eggs of the cattle tick Boophilus microplus. This proteinase, herein named THAP (tick heme-binding aspartic proteinase) showed pepstatin-sensitive hydrolytic activity against several peptide and protein substrates. Although hemoglobin was a good substrate for THAP, low proteolytic activity was observed against globin devoid of the heme prosthetic group. Hydrolysis of globin by THAP increased as increasing amounts of heme were added to globin, with maximum activation at a heme-to-globin 1:1 ratio. Further additions of heme to the reaction medium inhibited proteolysis, back to a level similar to that observed against globin alone. The addition of heme did not change THAP activity toward a synthetic peptide or against ribonuclease, a non-hemeprotein substrate. The major storage protein of tick eggs, vitellin (VT), the probable physiological substrate of THAP, is a hemeprotein. Hydrolysis of VT by THAP was also inhibited by the addition of heme to the incubation media. Taken together, our results suggest that THAP uses heme bound to VT as a docking site to increase specificity and regulate VT degradation according to heme availability.