Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide
2006; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês
10.1074/jbc.m610503200
ISSN1083-351X
AutoresTim J. Winterburn, David M. Wyatt, Lowri H. Phylip, Daniel Bur, Rebecca Harrison, Colin Berry, John Kay,
Tópico(s)Enzyme Production and Characterization
ResumoThe 68-residue IA3 polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2–32 of the polypeptide adopt an amphipathic α-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (Ki < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA3 from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA3. The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained. The 68-residue IA3 polypeptide from Saccharomyces cerevisiae is essentially unstructured. It inhibits its target aspartic proteinase through an unprecedented mechanism whereby residues 2–32 of the polypeptide adopt an amphipathic α-helical conformation upon contact with the active site of the enzyme. This potent inhibitor (Ki < 0.1 nm) appears to be specific for a single target proteinase, saccharopepsin. Mutagenesis of IA3 from S. cerevisiae and its ortholog from Saccharomyces castellii was coupled with quantitation of the interaction for each mutant polypeptide with saccharopepsin and closely related aspartic proteinases from Pichia pastoris and Aspergillus fumigatus. This identified the charged K18/D22 residues on the otherwise hydrophobic face of the amphipathic helix as key selectivity-determining residues within the inhibitor and implicated certain residues within saccharopepsin as being potentially crucial. Mutation of these amino acids established Ala-213 as the dominant specificity-governing feature in the proteinase. The side chain of Ala-213 in conjunction with valine 26 of the inhibitor marshals Tyr-189 of the enzyme precisely into a position in which its side-chain hydroxyl is interconnected via a series of water-mediated contacts to the key K18/D22 residues of the inhibitor. This extensive hydrogen bond network also connects K18/D22 directly to the catalytic Asp-32 and Tyr-75 residues of the enzyme, thus deadlocking the inhibitor in position. In most other aspartic proteinases, the amino acid at position 213 is a larger hydrophobic residue that prohibits this precise juxtaposition of residues and eliminates these enzymes as targets of IA3. The exquisite specificity exhibited by this inhibitor in its interaction with its cognate folding partner proteinase can thus be readily explained. Aspartic proteinases are widely distributed in nature and are involved in a variety of physiological processes and pathological conditions such as Alzheimer disease, cancer, hypertension, and AIDS (1Dunn B.M. Chem. Rev. 2002; 102: 4431-4458Crossref PubMed Scopus (296) Google Scholar). In stark contrast, naturally occurring protein inhibitors of aspartic proteinases are rare and are found only in a few specialized locations (1Dunn B.M. Chem. Rev. 2002; 102: 4431-4458Crossref PubMed Scopus (296) Google Scholar, 2Winther J.R. Phylip L.H. Kay J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier Academic Press, Inc., London2004: 87-90Crossref Scopus (4) Google Scholar, 3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). There is thus a need to understand the mechanism of action of the few inhibitors that do exist with a view to exploitation of their therapeutic potential. One such inhibitor is the IA3 polypeptide from Saccharomyces cerevisiae. This is a potent inhibitor (Ki < 10-10 m) of its target enzyme, the vacuolar proteinase from S. cerevisiae, known previously as proteinase A but which is now better denoted as saccharopepsin (2Winther J.R. Phylip L.H. Kay J. Barrett A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. 2nd Ed. Elsevier Academic Press, Inc., London2004: 87-90Crossref Scopus (4) Google Scholar). Remarkably, IA3 appears to be completely specific for this sole target proteinase (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). A wide variety of other aspartic proteinases of vertebrate, parasite, plant, and fungal origin are not only unaffected by IA3 but have been shown to digest it as a substrate (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Although IA3 consists of 68 residues, we have shown previously that all of the inhibitory activity resides within the N-terminal half of the polypeptide (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). By itself, free IA3 is essentially unstructured (5Green T. Ganesh O. Perry K. Smith L. Phylip L.H. Logan T.M. Hagen S.J. Dunn B.M. Edison A.S. Biochemistry. 2004; 45: 4071-4081Crossref Scopus (32) Google Scholar), but, upon contact with its target proteinase, the polypeptide operates as an inhibitor through an unprecedented mechanism (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Residues 2–32 become ordered and adopt an almost perfect amphipathic helical conformation occupying the active site of the enzyme (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). The hydrophilic face of the IA3 helix is oriented predominantly toward the solvent, whereas the face enclosed by the target enzyme is largely hydrophobic. Our previous biochemical and crystallographic investigations have revealed that the potency of the interaction of IA3 with saccharopepsin is mostly generated by the insertion of a series of hydrophobic amino acids into complementary hydrophobic pockets provided by the active site cleft of the enzyme (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Within the IA3 inhibitory sequence of residues 2–34 (Fig. 1), these consist of (i) an N-terminal cluster of V8-X- X-I11-F12, 3For ease of understanding, residues in the inhibitor(s) are denoted by single-letter abbreviations, whereas proteinase residues are indicated in the three-letter code.3For ease of understanding, residues in the inhibitor(s) are denoted by single-letter abbreviations, whereas proteinase residues are indicated in the three-letter code. (ii) a C-terminal cluster of V26-X-X-A29-F30, and (iii) the leucine residue at position 19. This is found within the sequence K18-L19-X-X-D22, which also contributes, somewhat unexpectedly, two charged amino acids, K18 and D22, in an i, i + 4 pair, to the hydrophobic face of the amphipathic helical inhibitor. The ε-NH+3 group of the K18 side chain is held in place through three hydrogen bonds, including one with the carboxyl group of Asp-32 from the enzyme and another with one of the carboxyl oxygens of the side chain of D22 (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). The other carboxyl oxygen of D22 is hydrogen-bonded to Tyr-75 contributed by the β-hairpin loop or "flap" that overlies the active site of the enzyme (4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). Through this intricate network of strong hydrogen bonds, K18 and D22 are tightly linked to each other and to crucial residues (Asp-32 and Tyr-75) of the enzyme's active site (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). However, since Asp-32 and Tyr-75 are totally conserved in the sequences of all active aspartic proteinases from eukaryotic sources, this arrangement cannot by itself account for the very high specificity observed in the interaction between IA3 and its cognate folding partner proteinase, saccharopepsin. Nevertheless, the K18 and D22 residues in the inhibitor were selected as the starting point for our investigations to identify the key features that facilitate this selective interaction because of their unusual positioning on the hydrophobic face of the helix, where they contribute little to the energetics of inhibitor binding. This was demonstrated recently by varying these residues considerably without diminishing the potency of the IA3 interaction with saccharopepsin at the standard pH of 4.7 employed throughout our studies (6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). Their influence on selectivity was established initially by mutagenesis of these residues and determination of the resultant effects on the inhibition not only of saccharopepsin but also of two fungal vacuolar aspartic proteinases (from Pichia pastoris and Aspergillus fumigatus) that are closely related to saccharopepsin (sequence identity ∼75%). Inspection of x-ray structures of IA3-inhibited saccharopepsin (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar) and models of these two other fungal proteinases identified, in turn, potential key residues for mutation within the saccharopepsin component of this remarkable proteinase/inhibitor partnership. DNA Manipulation and Production of Recombinant Proteinases—Mutations were introduced by overlapping PCR mutagenesis (7Tigue N.J. Kay J. J. Biol. Chem. 1998; 273: 26441-26446Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) into the wild-type saccharopepsin sequence to generate clones encoding the Thr222Ala, Thr287Met/Pro288Gly, and Ala213Ile mutant proteinases. The respective forward (F) and reverse (R) primers used were as follows: 222F, GATACTGGTACTTCTTTGATTGCATTGCCATCAGGATTAGCTG; 222R, CAGCTAATCCTGATGGCAATGCAATCAAAGAAGTACCAGTATC; 287/288F, CTCCTGTATCTCTGCAATTATGGGAATGGATTTCCCAGAAC; 287/288R, GTTCTGGGAAATCCATTCCCATAATTGCAGAGATACAGGAG; 213F, GAATTGGAGAGCCATGGTGCCATAATCGATACTGGTACTTCTTTGATTA; 213R, TAATCAAAGAAGTACCAGTATCGATTATGGCACCATGGCTCTCCAATTC. Each mutant DNA, ligated into the BamHI-XbaI sites of pESC-URA3 (Stratagene) was transformed into S. cerevisiae cells deficient in wild-type saccharopepsin (8van den Hazel H.B. Kielland-Brandt M.C. Winther J.R. Eur. J. Biochem. 1992; 207: 277-283Crossref PubMed Scopus (59) Google Scholar) and expressed as described previously for the wild-type enzyme (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Each mutant enzyme, as well as wild-type saccharopepsin, was purified from the respective conditioned medium, essentially as described previously for the wild-type enzyme (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). The gene encoding the proenzyme form of the vacuolar proteinase from P. pastoris does not contain any introns, so the coding sequence was amplified from P. pastoris genomic DNA by PCR using 5′-GTGATCAATGATATTTGACGGTACTACGATG-3′ and 5′-GGTCGACCTAAATAGACTTGGCTAAACCTAC-3′ as forward and reverse primers, respectively. Following cloning into the vector used for wild-type saccharopepsin, expression was performed in Saccharomyces cerevisiae cells deficient in saccharopepsin (8van den Hazel H.B. Kielland-Brandt M.C. Winther J.R. Eur. J. Biochem. 1992; 207: 277-283Crossref PubMed Scopus (59) Google Scholar). The recombinant Pichia proteinase was purified from the conditioned medium using the same protocol as that described previously for saccharopepsin (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Sequencing (through 12 cycles of Edman degradation) of the recombinant Pichia proteinase gave Ala-Ser-His-Asp-Ala-Pro-Leu-Thr-Asn-Tyr-Leu-Asn∼, which corresponds precisely to that of the mature form of the proteinase predicted by the DNA sequence. A clone encoding the precursor of the vacuolar aspartic proteinase from A. fumigatus in the pPICZαA vector was a kind gift from Professor M. Monod (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland). This was expressed in P. pastoris cells as described in Ref. 9Reichard U. Cole G.T. Rüchel R. Monod M. Int. J. Med. Microbiol. 2000; 290: 85-96Crossref PubMed Scopus (41) Google Scholar, and the recombinant proteinase was purified to homogeneity from the culture medium by a slight modification of the protocol originally described (9Reichard U. Cole G.T. Rüchel R. Monod M. Int. J. Med. Microbiol. 2000; 290: 85-96Crossref PubMed Scopus (41) Google Scholar). This was necessary to remove the yellow pigment that is produced in conditioned medium produced by P. pastoris cells. Briefly, this involved dialysis of the culture medium for 72 h against 10 mm sodium formate buffer, pH 4.0, containing 1 mm EDTA, followed by successive concentrations in a stirred cell concentrator (Amicon, Beverly, MA) fitted with a 10-kDa cut-off membrane and then in a Vivaspin concentrator (Vivascience Sartorius Ltd., Epsom, UK) fitted with a 5-kDa cut-off membrane. The concentrate was subjected to successive chromatographies on Sephadex G-50 and a Resource Mono S column (Amersham Biosciences) in the same formate buffer. Elution from the ion exchange column was achieved with a 0–50% gradient of the formate buffer containing 1 m NaCl. The purified Aspergillus enzyme was found to have microheterogeneity at its N terminus, as described in the original report on the production of this recombinant enzyme (9Reichard U. Cole G.T. Rüchel R. Monod M. Int. J. Med. Microbiol. 2000; 290: 85-96Crossref PubMed Scopus (41) Google Scholar). DNA Manipulations and Production of Recombinant Inhibitors—Mutations were introduced, simultaneously where appropriate, at residues 18 and 22 to produce some mutant forms of S. cerevisiae IA3 (inhibitors 2–11, Tables 2 and 3), as described previously (6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). The QuikChange kit (Stratagene) or overlapping PCR methods (7Tigue N.J. Kay J. J. Biol. Chem. 1998; 273: 26441-26446Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) were used to introduce the mutations, using the primer pairs as detailed in supplemental Table 1. Other mutant inhibitors (inhibitors 16–18 described in Table 4 and 25–27 in Table 6) were produced by engineering a cassette version of the DNA encoding S. cerevisiae IA3. An unwanted SacI site near the 3′-end of the IA3 sequence was removed, and an NheI site was introduced as a silent mutation in the codons for Ala-34-Ser-35 (GCT AGT → GCT AGC) by (separate) site-directed mutageneses using QuikChange. Digestion with SacI/NheI enabled removal of the bases encoding wild-type residues 15–34. Replacement with appropriate pairs of synthetic oligonucleotides carrying the desired base changes enabled the mutations to be introduced; the oligonucleotide pairs used for these mutant forms of IA3 are shown in supplemental Table 1.TABLE 2Interaction of the vacuolar proteinases from S. cerevisiae (saccharopepsin), Pichia pastoris (PpPr), and Aspergillus fumigatus (AfPr) with recombinant wild-type S. cerevisiae IA3 and mutant forms containing hydrophobic residues at positions 18, 19, and 22 Inhibition constants were derived at the standard pH of 4.7 for all three enzymes and, additionally, at pH 3.1 for saccharopepsin. NI, no inhibition at 1 μm.IdentityResidue at positionSaccharopepsin KiPpPr Ki pH 4.7AfPr Ki pH 4.7181922pH 3.1pH 4.7nmnmnm1KLD1.1 ± 0.4<0.155 ± 11NI2MLL<0.1<0.10.5 ± 0.535 ± 53ILI<0.1<0.1<0.19 ± 14IMI2 ± 0.3<0.10.6 ± 0.1513 ± 2 Open table in a new tab TABLE 3Interaction at pH 4.7 of the vacuolar proteinases from S. cerevisiae (saccharopepsin) and P. pastoris (PpPr) with recombinant forms of S. cerevisiae IA3 and the protein from S. castellii, with variant residues at positions 18 and 22 Inhibitors 5–11 consisted of the sequence of S. cerevisiae IA3, whereas inhibitors 12–15 had the sequence of the protein from S. castellii (Fig. 1), with the indicated residues at positions 18 and 22 in each case. The ratio of the Ki for each inhibitor against PpPr relative to that for saccharopepsin is indicated.IdentityResidue at positionSaccharopepsin KiPpPr KiRatio1822nmnm5KM 1506RD 607DK0.3 ± 0.06aData taken from Ref. 6300 ± 501,0008DR5 ± 0.8>500bIC50 value>1009ER0.7 ± 0.3160 ± 1523010VK2 ± 0.4aData taken from Ref. 635 ± 21711MK0.2 ± 0.04aData taken from Ref. 63 ± 0.91512MK4 ± 0.515 ± 3∼413KM370 ± 50>500bIC50 value∼114KL1,000 ± 200>800bIC50 value∼115KD3 ± 0.810 ± 5∼4a Data taken from Ref. 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholarb IC50 value Open table in a new tab TABLE 4Inhibition at pH 4.7 of the vacuolar proteinases from S. cerevisiae (saccharopepsin) and P. pastoris (PpPr) by variant forms of S. cerevisiae IA3 with residues at the indicated positions replaced by their S. castellii counterparts Residues in boldface type are derived from the S. castellii sequence. The ratio is defined as in the legend to Table 3. Open table in a new tab TABLE 6Interactions at pH 4.7 between wild-type and Ala213Ile mutant forms of saccharopepsin and variant forms of IA3 Inhibitors 21–24 were synthetic peptides consisting solely of the residues shown. In these forms of IA3, l-norleucine (Z) was substituted for methionine. Inhibitors 11, 18, and 25–27 were recombinant proteins consisting of the sequences shown fused to residues 35–68 from S. cerevisiae IA3 (Fig. 1). Residues from the S. castellii sequence that differ from those in S. cerevisiae IA3 are highlighted in boldface type. The ratio for the Ki for each inhibitor against the Ala213Ile mutant relative to that for wild-type saccharopepsin is indicated. Open table in a new tab Genomic DNA was extracted from S. castellii, and the gene encoding the inhibitor ortholog was amplified by PCR using 5′-CATATGAGTGATAAAAACGCTAACG-3′ and 5′-CTCGAGATGATCCATCAATTCATCTTTATC-3′ as the forward and reverse primers, respectively. Mutations were introduced into this wild-type sequence to generate the M18K/K22M, M18K/K22L, and M18K/K22D variants (inhibitors 13–15 respectively, Table 3) using the primers and methods indicated in supplemental Table 1. Wild-type and mutant forms of IA3 from S. cerevisiae and S. castellii were subcloned into the NdeI-XhoI sites of pET-22b (Novagen, Milton Keynes, UK), thus introducing a C-terminal Leu-Glu-His6 tag as described previously (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). All recombinant protein forms were produced in Escherichia coli and purified to homogeneity by nickel-chelate chromatography, as reported previously (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). Synthetic peptide forms (inhibitors 19–24) consisting of residues 2–32 or 2–34 from the S. cerevisiae or S. castellii sequences (Fig. 1) were obtained from Alta Biosciences (Birmingham, UK) and had l-norleucine residues introduced in place of methionine, where appropriate, as discussed previously (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). Inhibition assays were carried out predominantly at pH 4.7, but in some cases, when the Ki value at this pH lay at or beyond the limit of accurate determination using the assay methodology available, it was necessary to make measurements at an alternative pH; the lower pH of 3.1 was used in these instances, as described previously (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). The synthetic chromogenic substrate (from Alta Bioscience) used in all assays was Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu (where the asterisk represents the scissile peptide bond, and Nph represents l-nitrophenylalanine (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar)). N-terminal sequencing was performed by automated Edman degradation (Alta Bioscience). Modeling calculations were carried out on an SGI Octane workstation with dual R12000 processors, using the Moloc program, as reported previously (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). Interaction of Variant Forms of IA3 with Fungal Proteinases— Two fungal aspartic proteinases that are closely related to saccharopepsin were identified by searching the sequence data bases. These enzymes, from P. pastoris (PpPr) 4The abbreviations used are: PpPr, vacuolar aspartic proteinase from P. pastoris; AfPr, vacuolar aspartic proteinase from A. fumigatus. and A. fumigatus (AfPr), have 77 and 71% identity, respectively, with the sequence of saccharopepsin and, in this regard, are more closely related to saccharopepsin than any of the aspartic proteinases that we showed previously to be unaffected by IA3 as an inhibitor (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar). PpPr and AfPr were produced in recombinant form as described under "Experimental Procedures," and kinetic parameters were determined for hydrolysis by these two proteinases of the synthetic peptide substrate that we have employed throughout all of our studies with IA3 (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 4Li M. Phylip L.H. Lees W.E. Winther J.R. Dunn B.M. Wlodawer A. Kay J. Gustchina A. Nat. Struct. Biol. 2000; 7: 113-117Crossref PubMed Scopus (85) Google Scholar, 6Winterburn T.J. Wyatt D.M. Phylip L.H. Berry C. Bur D. Kay J. Biol. Chem. 2006; 387: 1139-1142Crossref PubMed Scopus (6) Google Scholar). The values obtained (Table 1) were comparable with those determined previously for wild-type saccharopepsin (3Phylip L.H. Lees W.E. Brownsey B.G. Bur D. Dunn B.M. Winther J.R. Gustchina A. Li M. Copeland T. Wlodawer A. Kay J. J. Biol. Chem. 2001; 276: 2023-2030Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Despite these similar abilities to interact with substrate and the high overall sequence identity between PpPr, AfPr, and saccharopepsin, the Pichia enzyme was only marginally affected at the standard pH of 4.7 (see "Experimental Procedures") by recombinant IA3 from S. cerevisiae (inhibitor 1 in Table 2); the Aspergillus enzyme was not inhibited at all. These findings with two fungal enzymes that are so closely related to saccharopepsin provide yet further substantiation of the high specificity of thi
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