[1]The Journal of Biological Chemistry [2]Skip to main page content * [3]Home * [4]Current issue * [5]Archive * [6]Papers in Press * [7]Minireviews * [8]Classics * [9]Reflections * [10]Papers of the Week QUICK SEARCHAuthor: _________Keyword: ________________Year: ____Vol: ____Page: ____ GO Go[11][Advanced Search][12][Browse the Archive] * Institution: Univ Colorado - Denison Memorial Library * [13]Sign In Advertisement Advertisement * [14]Celebrate 100 Years of ASBMB: ASBMB Histroy Book Now Avaiable! 1 The Potency and Specificity of the Interaction between the IA[3] Inhibitor and Its Target Aspartic Proteinase fromSaccharomyces cerevisiae[15]* 1 1. [16]Lowri H. Phylip[17]tOa, 2. [18]Wendy E. Lees[19]tOa[20]FNb, 3. [21]Brian G. Brownsey[22]tOc, 4. [23]Daniel Bur[24]tOd, 5. [25]Ben M. Dunn[26]tOe, 6. [27]Jakob R. Winther[28]tOf, 7. [29]Alla Gustchina[30]tOg, 8. [31]Mi Li[32]tOg[33]tOh, 9. [34]Terry Copeland[35]i, 10. [36]Alexander Wlodawer[37]tOg and 11. [38]John Kay[39]tOa[40]FNj 1. From the ^tOaSchool of Biosciences, Cardiff University, P. O. Box 911, Cardiff CF10 3US, Wales, United Kingdom, the^tOcDepartment of Medicine, University of Wales College of Medicine, Cardiff CF14 4XN, Wales, United Kingdom, ^tOdF. Hoffmann La Roche AG, CH-4070 Basel, Switzerland, the ^tOeDepartment of Biochemistry & Molecular Biology, University of Florida College of Medicine, Gainesville, Florida 32610, the ^tOfDepartment of Yeast Genetics, Carlsberg Laboratory, DK-2500, Copenhagen Valby, Denmark, the^tOgProtein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, Maryland 21702, the^tOhIntramural Research Support Program, SAIC Frederick, National Cancer Institute, Frederick, Maryland 21702, and the ^iProgram Core, DBS, National Cancer Institute, Frederick, Maryland 21702 [41]Next Section 2 Abstract 2 The yeast IA[3] polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA[3] 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 IA[3] polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequence. Crystal structures of different forms of IA[3] complexed with proteinase A showed that residues in the N-terminal half of the IA[3] sequence became ordered and formed an almost perfect α-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 IA[3] was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA[3] 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. Aspartic proteinases participate in a variety of physiological processes, and the onset of pathological conditions such as hypertension, gastric ulcers, and neoplastic diseases may be related to changes in the levels of their activity. Members of this proteinase family, e.g. renin, pepsin, cathepsin D, and human immunodeficiency virus-proteinase are generally type-cast on the basis of their susceptibility to inhibition by naturally occurring, small molecule inhibitors such as the acylated pentapeptides, isovaleryl- and acetyl-pepstatin. However, the two most recently identified human aspartic proteinases, β-site Alzheimer's precursor protein cleavage enzyme and β-site Alzheimer's precursor protein cleavage enzyme 2 ([42]1, [43]2), are not inhibited by this classical type of inhibitor of this family of enzymes. Pepstatins are metabolic products produced by various species of actinomycetes and, as such, are not themselves gene-encoded. Protein inhibitors of aspartic proteinases are relatively uncommon and are found in only a few specialized locations ([44]3). Examples include renin-binding protein in mammalian kidneys which intriguingly has now itself been identified to be the enzyme,N-acetyl-d-glucosamine-2-epimerase ([45]4); a 17-kDa inhibitor of pepsin and cathepsin E from the parasite, Ascaris lumbricoides ([46]5); proteins from plants such as potato, tomato, and squash ([47]6, [48]7), and a pluripotent inhibitor from sea anemone of cysteine proteinases as well as cathepsin D ([49]8). The IA[3] polypeptide in yeast is an 8-kDa inhibitor of the vacuolar aspartic proteinase (proteinase A or saccharopepsin) that was initially described by Holzer and co-workers ([50]9). The complete sequence of this 68-residue inhibitor has been elucidated ([51]10, [52]11) and the inhibitory activity of IA[3] has been shown to reside within the N-terminal half of the molecule ([53]10, [54]12). We have recently solved the structure of the IA[3]-proteinase A complex ([55]12), demonstrating that whereas free IA[3] has little intrinsic secondary structure, residues 232 of the inhibitor, upon contact with proteinase A, become ordered and adopt a near perfect α-helical conformation occupying the active site cleft of the enzyme. This was the first crystal structure to be determined for a gene-encoded aspartic proteinase inhibitor complexed with its target enzyme. It was thus considered important to investigate further the role of the proteinase as a folding template and to attempt to establish the molecular features that enable this unprecedented mode of inhibitor-proteinase interaction to occur. [56]Previous Section[57]Next Section 2 EXPERIMENTAL PROCEDURES 2 5 Protein Production and Purification 5 Proteinase A and other aspartic proteinases were obtained, and peptides were synthesized by solid-phase methods, as described previously ([58]12). Genomic DNA was extracted from S. cerevisiae and the gene encoding IA3 was amplified specifically by polymerase chain reaction (PCR)^1 using 5′-GCATATGAATACAGACCAACAAAAAGTG-3′ and 5′-GCTCGAGCTCCTTCTTATGCCCCGC-3′ as forward and reverse primers. Mutations were introduced into the wild type sequence to generate clones encoding the chimera, (Gly)[9] and K7M proteins (Table[59]I) by using the respective forward primers: chimera, 5′-GGAGATATACATATGGGAGGACACGACGTCCCTTTAACAAACATATTTCAGAGCTCA-3′; (Gly)[9], 5′-GCATATGGGAGGAGGCGGCGGCGGTGGAGGAGGCATATTTCAGAGCTCA-3′; and K7M, 5′-GCATATGAATACAGACCAACAAATGGTGAGCGAA-3′ in conjunction with the wild type reverse primer described above. The constructs encoding the K24M, K31M/K32M, Mix, D22L, and K18M/D22L mutants were each generated in two steps by overlapping PCR mutagenesis ([60]13) using the mutagenic primer sets: K24M, forward, 5′-GGCGATGCAATGGTAGTGAGTGACGCTTTT-3′ and reverse, 5′-ACTCACTACCATTGCATCGCCCTGCAATTT-3′; K31M/ K32M: forward, 5′-TTTATGATGATGGCCAGTCAAGACAAGGACGGC-3′ and reverse 5′-ACTGGCCATCATCATAAAAGCGTCACTCACTACCTT-3′; Mix: forward, 5′-AAGGCCGATAAATTTTCAATGGCTAGTCAAGACAAGG-3′ and reverse, 5′-TGAAAATTTATCGGCCTTCACTACCTTTGCATCGCC-3′; D22L: forward, 5′-CAGGGGCTGGCCAAGGTAGTGAGTGACGCTTTT-3′ and reverse, 5′-TACCTTGGCCAGCCCCTGCAACTTTTCCTTTGA-3′; K18M/D22L: forward, 5′-GAAATGTTGCAGGGGCTGGCCAAGGTAGTGAGTGACGCTTTT-3′ and reverse, 5′-ATCCTTGGCCAGCCCCTGCAACATTTCCTTTGAGCTCTGAAA-3′. The second set of overlap primers utilized the T7 forward primer and the wild type reverse primer described above. View this table: * [61]In this window * [62]In a new window Table I Interactions between yeast proteinase A and protein forms of IA[3] Wild type and mutant forms of IA3 were subcloned into theNdeI-XhoI sites of pET-22b ([63]Novagen, Cambridge, United Kingdom), thus introducing a C-terminal Leu-Glu-His[6]tag. Escherichia coli strain BL21DE3(pLysS) was transformed with wild type or mutant clones, then grown at 37 C in LB medium to an A [600] of ∼0.6 before induction with 1 mmisopropyl-1-thio-β-d-galactopyranoside. Each soluble recombinant protein was loaded onto a nickel-chelate affinity column in 0.05 m sodium phosphate buffer, pH 8.0, containing 0.3 m NaCl, washed with the same buffer adjusted to pH 6.0 containing 10% glycerol and each protein form of IA[3] was eluted with the glycerol containing buffer at pH 4.0. Appropriate fractions containing IA[3] were then heated twice at 100 C for 5 min. 5 Analytical Measurements 5 N-terminal sequencing of wild-type and mutant forms of IA[3] was performed by automated Edman degradation on protein bands that were electroblotted onto polyvinylidene difluoride membrane following SDS-polyacrylamide gel electrophoresis on 20% gels. Samples for amino acid analysis were hydrolyzed for 16 h at 105 C in 6 m HCl before being loaded onto a Biochrom 20 amino acid analyzer (Amersham Pharmacia Biotech, Cambridge, UK). MALDI-TOF mass spectrometry was carried out using a PE Biosystems Voyager Elite XL instrument incorporating a UV laser and delayed extraction. Samples at ∼10 pmol/μl were added to a matrix solution consisting of ferulic acid (55 mg/ml in ethanol containing 0.1% trifluoroacetic acid) in a sample/matrix ratio of 3:1. 5 Kinetic Measurements and Peptide Cleavage 5 Inhibition assays were conducted at pH 3.1 and 4.7 as described previously ([64]12, [65]14) using Lys-Pro-Ile-Glu-Phe*NitroPhe-Arg-Leu (where the asterisk indicates the scissile peptide bond) as substrate for all enzymes except yapsin 1 ([66]15). For this enzyme, the substrate used was ACTH (residues 139). This was incubated at 37 C in 50 mm sodium acetate buffer, pH 5.3, with 10 units of purified yapsin 1 ([67]15) for 30 min in the absence and presence of peptide 1 at final concentrations up to 20 μm. The proteolytic product from ACTH (residues 115) was detected and quantified by reverse phase high performance liquid chromatography. The susceptibility of peptide/protein forms of IA[3] to proteolytic cleavage was examined by incubation at 37 C for various lengths of time (commonly 16 or 72 h) with the appropriate enzyme in 100 mm sodium formate/acetate buffers at pH 3.1/4.7, respectively, each containing 300 mm NaCl. Each synthetic peptide was incubated initially with proteinase A for 16 h at a molar ratio of 40:1 and, if no cleavage was detected under these circumstances, then the incubation was repeated at a peptide:proteinase ratio of 10:1 for 72 h. Peptide 1 was incubated with nontarget proteinases such as human pepsin at both pH 3 and 5 at a molar ratio of 1,000:1 for only 1 h. Each digest was separated by reverse-phase fast protein liquid chromatography using a Pep-RPC column (Amersham Pharmacia Biotech, Bucks, United Kingdom) and the fractions containing any cleavage products were collected and subjected to acid hydrolysis followed by amino acid analysis. Protein samples were frozen for storage prior to subsequent mixing with the matrix solution for application to a stainless steel target and analysis by MALDI-TOF mass spectrometry. 5 Crystallography and Molecular Modeling 5 Crystals of a complex of proteinase A with the K24M mutant protein form of IA[3] (Table [68]I) were grown by vapor diffusion under the conditions described previously for other IA[3]-proteinase A complexes ([69]12). The initial solution was prepared at a molar ratio of inhibitor:proteinase of 5:1, and after separation from the excess of inhibitor by gel filtration on Sephadex G-50, was concentrated to 5 mg/ml by ultrafiltration. The mother liquor contained 30% PEG1500, 0.14 m ammonium sulfate in 0.1 m MES buffer, pH 6.0. Data extending to 1.9 were collected at 100 K on beamline X9B at NSLS, Brookhaven National Laboratory, Upton, NY, using an ADSC 4K CCD detector. Data were processed with HKL2000 ([70]16). The initial data set consisted of 217,446 reflections that could be scaled withR [sym] of 8.7% (last shell 34.6%) to yield 41,718 unique measurements. The completeness was 92.8% for the whole data and 75.5% for the final shell. The structure of proteinase A complexed with peptide 1 ([71]12) was used as the initial model with replacement of Lys^24 by Met. The structure was refined with CNS 1.0 ([72]17) utilizing data extending to 2.0- resolution. The first two rounds of refinement included positional and B factor refinements and model adjustment, while solvent molecules were added in the third round. The final model contained the enzyme, residues 231 of the inhibitor and 243 water molecules. The final R factor was 19.84% and R [free] was 23.1%. The root mean square deviations for bond lengths and bond angles from ideality was 0.012 and 1.59 , respectively. Modeling calculations were made on a Silicon Graphics Octane with a single R12000 processor using the Moloc modeling package. Individual amino acid side chains (K18M/D22L) in IA[3] were changed with a built-in function in Moloc. Side chain conformations were adapted manually and a subsequent round of optimization, maintaining proteinase A and the remainder of IA[3] fixed, resulted in low energy conformations for the newly introduced side chains. The new protein-inhibitor complex was checked for attractive and repulsive interactions, and allowed conformations, respectively. [73]Previous Section[74]Next Section 2 RESULTS AND DISCUSSION 2 5 Interaction of Protein/Peptide Forms of IA[3] with Target and Nontarget Enzymes 5 The nucleotide sequence encoding all 68 residues of IA[3] was amplified by PCR and introduced into the pET-22b vector for expression in E. coli as described under Experimental Procedures. The recombinant protein that accumulated in E. coli was soluble and was purified to homogeneity from cell lysates by taking advantage of the His[6] tag introduced from the pET-22b vector at the C terminus of the IA[3] polypeptide chain (see Experimental Procedures). N-terminal analysis of one batch of the homogeneous wild-type inhibitor through 10 cycles of Edman degradation gave the sequence Asn-Thr-Asp-Gln-Gln-Lys-Val-Ser-Glu-Ile, which is exactly coincident with that predicted by the DNA sequence for residues 211 (Table [75]I) ([76]11) and indicates that the initiator Met^1 residue had been removed during the accumulation of this batch of recombinant protein in E. coli. Analysis of a separate batch of recombinant wild-type inhibitor by MALDI-TOF mass spectometry gave a mass of 8772 Da, identical with that predicted (8772 Da) for the IA[3]sequence plus the C-terminal extension of ∼Leu-Glu-His[6]introduced from the pET-vector. The K [i] value determined at pH 3.1 for the inhibition of yeast proteinase A by this C-terminal tagged, wild-type recombinant protein (wild-type in Table [77]I) was comparable to that reported previously at the same pH for the naturally occurring protein purified from S. cerevisiae ([78]14). It is readily apparent then that the introduction of the extra ∼Leu-Glu-His[6] residues at the C terminus of the recombinant inhibitor did not have any significant detrimental effect on inhibitory potency. Since the target proteinase is unlikely ever to encounter a pH as low as 3.1 in its cellular environment, attempts were also made to determine inhibition constants at higher pH values such as 4.7 and 6.0. In both cases, the interaction with proteinase A was so tight that theK [i] values lay at or beyond the limits of accurate determination using the available assay methodology and so were estimated to be <0.1 nm. A synthetic peptide which spanned residues 234 of the IA[3]sequence (Peptide 1 in Table [79]II) was found to have inhibitory potency against yeast proteinase A at pH 3.1 and 4.7 comparable to those of the naturally occurring and wild-type recombinant protein forms of IA[3], described previously ([80]12,[81]14). In contrast, peptide 1, at a concentration of 2 μm, had no significant ability to inhibit any one of a number of other aspartic proteinases from a wide range of other species (Table[82]III). These have considerable sequence and structural similarities to yeast proteinase A and included yapsin 1 (a membrane-attached aspartic proteinase also from S. cerevisiae ([83]15) and other enzymes of fungal, mammalian, parasite (plasmepsin II from Plasmodium falciparum ([84]18) and plant (cyprosin from Cynara cardunculus ([85]19)) origin. Thus, IA[3] is a potent specific inhibitor directed solely against its target enzyme, yeast proteinase A. Since peptide 1 had no effect on the nontarget enzymes listed in Table [86]III, reciprocally, the effect of a number of these (including e.g. human pepsin, cathepsin D, and cathepsin E) on peptide 1 was examined. With human pepsin, for example, using a molar ratio of peptide 1:pepsin of 1,000:1 at pH 3.1 and pH 5, peptide 1 was cleaved rapidly at the ∼Glu^10* Ile^11∼ and ∼Ala^29*Phe^30∼ bonds, as revealed by amino acid analysis of the collected peptide fragments (data not shown). Identical results were obtained for the other enzymes so it would appear that peptide 1 is unable to inhibit the nontarget aspartic proteinases such as pepsin, cathepsins D and E (and the other enzymes listed in Table [87]III) because these enzymes cut the polypeptide effectively as a substrate. Consequently, the ∼Ala^29*Phe^30∼ and ∼Glu^10*Ile^11∼ sites that were cleaved by the nontarget proteinases were changed singly and in concert to introduce residues at the P[1] position which were known from extensive previous studies (e.g. Refs. [88]20 and [89]21) to be refractory to cleavage by such enzymes as human pepsin and cathepsin D. The resultant peptides (2 and 3, Table [90]II) were just as potent as peptide 1 as inhibitors of proteinase A but still showed no significant ability to inhibit, e.g. human pepsin or cathepsin D at concentrations as high as 5 μm. Although cleavage of peptides 2 and 3 between residues ∼Val^29-Phe^30∼ and ∼Lys^10-Ile^11∼ plus ∼Val^29-Phe^30∼, respectively, was no longer evident, nevertheless, cleavage at other locations now became apparent. For example, after incubation of peptide 3 with human cathepsin D at pH 3.1, the digest was analyzed by MALDI-TOF mass spectrometry and the large product that was detected (3238 Da observed; 3237 Da predicted) indicated that cleavage had occurred between residues ∼Gln^5*Gln^6∼ to generate the fragment spanning residues 634. Thus, peptides 1, 2, and 3 appeared to be cleaved by the nontarget proteinases including human pepsin and cathepsin D at whatever peptide bonds were accessible and which met the sub-site specificity requirements of each enzyme. View this table: * [91]In this window * [92]In a new window Table II Interactions between yeast proteinase A and synthetic peptide forms of IA[3] View this table: * [93]In this window * [94]In a new window Table III Aspartic proteinases unaffected by the IA[3] inhibitor In contrast, peptides 1, 2, and 3 were not cleaved by proteinase A (at pH 3.1 or 4.7), even upon prolonged incubation for 3 days at 37 C at molar ratios of peptide:proteinase A as low as 5:1. Similarly, the recombinant, wild-type protein form of IA[3] (Table [95]I) was not cleaved by yeast proteinase A since the mass ion (8772 Da) observed by MALDI-TOF mass spectrometry after prolonged incubation was identical to that of the starting material. However, the wild-type protein was digested by pepsin and cathepsin D, e.g. analysis of the material incubated at pH 3.1 with human cathepsin D indicated that one large product had accumulated which was consistent in size (5575 Da observed; 5578 predicted) with that of a fragment spanning residues Phe^30-Glu^68 plus the ∼Leu-Glu-(His)[6] tag. Thus the full-length protein form of IA[3] also appears to be degraded by nontarget proteinases with cleavage taking place, at least, at one of the bonds (∼Ala^29*Phe^30∼) that was identified earlier for peptide 1 (no attempts were made to detect by mass spectrometry any small products from the N terminus of IA[3] in the cathepsin D digest). This ready susceptibility of IA[3] in both peptide and protein forms to proteolytic cleavage by nontarget enzymes provides further substantiation to our conclusion described previously ([96]12) that the free IA[3] polypeptide has little intrinsic secondary structure. 5 Truncation and Mutagenesis of IA[3] 5 Since the inhibitory activity of IA[3] toward proteinase A appeared to be contained within residues 234 (compare K [i]values for the wild-type protein form (Table [97]I) and the peptide form (peptide 1, Table [98]II)), the effect on inhibitory potency of further truncation of this sequence was examined using a systematic series of synthetic peptides in which residues were successively deleted from the N and C termini. Removal of Asn^2 had little effect on theK [i] value measured at pH 3.1 (compare peptide 4 with peptide 1, Table [99]II) but the potency was diminished somewhat at pH 4.7, as a K [i] value was readily quantified for peptide 4 at this pH value (Table [100]II). Similarly, deletion of residues 36 progressively diminished inhibitory potency (peptides 5 and 6, Table[101]II) and the absence of residues 211 (in peptide 7) resulted in almost complete abolition of the inhibitory activity. The activity of IA[3] was totally destroyed when residues 215 were lacking (peptide 8, Table [102]II). The residues at the N-terminal end of the 234 polypeptide sequence would thus appear to contribute substantially to the potency of inhibition. To investigate the importance of the individual side chains of these residues, alterations in sequence were introduced into the full-length protein form of IA[3] at its N terminus by PCR mutagenesis as described under Experimental Procedures. Initially, residues 210 of the wild-type IA[3] sequence were replaced with Gly-Gly-His-Asp-Val-Pro-Leu-Thr-Asn. This is the sequence of residues that is present at the N terminus of the target enzyme, yeast proteinase A itself ([103]22) and, as such, was chosen totally arbitrarily. The recombinant, chimera inhibitor was purified to homogeneity and 10 cycles of Edman degradation yielded the sequence Gly-Gly-His-Asp-Val-Pro-Leu-Thr-Asn-Ile, identical to that predicted by the DNA sequence, and indicating once again that the initiator Met^1 residue had been removed by E. coli proteinases. Consistent with this, analysis by MALDI-TOF mass spectrometry gave a mass of 8502 Da, identical to that (8502 Da) predicted for the sequence of residues 268 plus the ∼Leu-Glu-His[6] tag of the chimeric protein. Remarkably, this chimeric protein with 9/34 (26%) of its residues exchanged was still almost as effective as an inhibitor as the wild-type protein, with its interaction at pH 4.7 still being so tight as to lie at or beyond the limits of accurate determination (Table [104]I). This result, together with the deletion experiments described above with the peptide form of IA[3], suggests that, for effective inhibition, backbone atoms contributed by residues 210 are essential but that the (side chain) identity of the individual amino acids in these positions is of lesser importance. On this basis, we replaced residues 210 of the natural sequence with nine glycine residues ((Gly)[9]mutant, Table [105]I) and purified the resultant protein to homogeneity. No attempts were made to sequence this protein because of the plethora of glycine residues but MALDI-TOF mass spectrometry gave a mass of 8124 Da, identical to that (8124 Da) predicted by the nucleotide sequence but, once again, lacking the initiator Met^1 residue. The yield of this mutant protein obtained from E. coli was about 5-fold lower than that obtained for the wild-type (and other mutant) sequence(s). This drastic introduction of nine consecutive glycine residues resulted in a poorer inhibitor with a K [i] of 40 nm at pH 3.1 (Table [106]I). However, at pH 4.7, the (Gly)[9] mutant protein was still a very effective inhibitor, with its potency quantified at around 1 nm (Table [107]I). Thus, the main chain atoms at the N terminus of the 234 sequence would appear to be the major contributors to inhibitory potency from this region with only minor influences being introduced by the individual residue side chains. This was substantiated further by replacement of individual residues, for example, the Lys^7 residue was replaced with methionine which is quasi-isosteric with lysine but lacks the ε-NH[2] group. This K7M mutant inhibitor was just as potent as the wild-type protein (Table [108]I). Truncation of the inhibitory sequence of residues 234 at its C terminus also resulted in a progressive loss of inhibitory potency (compare peptide 1 and peptides 9, 10, 11, 12, and 13, Table [109]II). Replacement of Lys^24 by Met (K24M mutant) and Lys^31 + Lys^32 (together) in the double mutant K31M/K32M again had no significant effect on the inhibitory potency of the resultant inhibitors toward proteinase A (Table [110]I). The structure of the K24M mutant complexed with proteinase A was solved at 2- resolution and refined to an R factor of 19.84% (see Experimental Procedures). Comparison with the structures reported previously ([111]12) for the K31M/K32M protein form and for the peptide 1 form of IA[3] complexed with proteinase A (Protein Data Bank accession codes [112]1dp5 and [113]1dpj) revealed that, in all three cases, residues 232 of the inhibitor had adopted a near-perfect α−helical conformation in the active site cleft of the enzyme. Electron density was only observed for these residues in all 3 structures and the root mean square deviation between the Cα coordinates was 0.224 between the protein and peptide form(s) of the inhibitor. The IA[3]helix is amphipathic with the charged residues including Lys^24 located on one face, protruding into solvent (Fig.[114]1). [115]Figure 1 View larger version: * [116]In this window * [117]In a new window * [118]Download as PowerPoint Slide Figure 1 The helical conformation adopted by residues 232 of IA[3] upon interaction with the active site of proteinase A. The sequence shown is that of the K31M/K32M mutant, depicting the distribution of selected hydrophilic and hydrophobic residues on opposite faces of the amphipathic helix. The structures all reveal that the main chain carbonyl and amido moieties of residues 210 of the IA[3] polypeptide are involved in H-bond formation with one another within the helix of the inhibitor but the side chains of these residues make no significant contacts with the proteinase, with the exception of Val^8which is involved in hydrophobic interactions (described later). In the sequence of the chimera inhibitor described earlier (Table [119]I), Val^8 was replaced with Leu which may be able to make hydrophobic contacts with the enzyme requiring only minor re-adjustment so that the derived K [i] value was not significantly perturbed. The binding energy of these contacts is clearly lost in the (Gly)[9] mutant protein but since the side chains of all of the other residues in the 210 sequence point largely into solvent, the still considerable inhibitory potency of the (Gly)[9]mutant (Table [120]I) can be readily understood. The predominant requirement in this N-terminal region appears to be for the backbone atoms to satisfy the H-bonding arrangement within the inhibitory helix, thereby stabilizing the helix by providing a somewhat lengthy cap. A comparable extended capping arrangement exists at the C-terminal end to stabilize the inhibitory helix. No electron density was observed for the side chains of any residues beyond Lys^32 in the crystal structure of the K24M mutant complex or in the structure of peptide 1 complexed with proteinase A described previously ([121]12) and the side chain of residue 32 makes no significant contacts with the enzyme. Yet the most potent inhibition (subnanomolar at pH 4.7) was measured when the IA[3] sequence was extended at its C terminus beyond Lys^32. This was achieved by the inclusion of Nle^33 (a synthetic isostere of the natural Met residue at this location) and Ala^34 in peptide 1 (Table [122]II) or by introducing a C-terminal Lys^32 amide (peptide 9-NH[2], Table [123]II) to stabilize the part of the inhibitor helix necessary for interaction with the proteinase by dissipation of the negative charge from the macromolecular dipole. The contribution of the main chain amido groups in satisfying the H-bonding arrangements at this end of the helix is substantiated by the increased potencies that were measured when each peptide terminated in a C-terminal amide instead of the free COOH group (compare peptides 9-NH[2], 10-NH[2] and 11-NH[2] with 9, 10, and 11, respectively, Table [124]I). A peptide that consisted only of residues 228 had lost essentially all of its inhibitory potency (peptide 12, Table[125]II) and residues 226 (peptide 13) were not active at all as an inhibitor. 5 Hydrophobic Clusters 5 Peptide 14 and peptide 8 (Table [126]II) together span the entire sequence of residues 234. Neither of these half-sized peptides was able to inhibit proteinase A when added singly or in combination with each other at a variety of molar ratios. Thus a contiguous sequence is necessary for IA[3] to inhibit proteinase A by forming the amphipathic helix (Fig. [127]1). The residues located on the hydrophobic face make extensive hydrophobic contacts with cognate residues in proteinase A. Particularly noticeable in this regard (Fig. [128]1) is the cluster arrangement of Val^8-X-X-hydrophobic-Phe^12toward the front end of the helix and Val^26-X-X-hydrophobic-Phe^30at the back end of the inhibitory sequence. In the front end cluster, replacement of Val^8 or Ile^11 individually by Ala had minor effects on inhibitory potency (compare peptides 15 and 16 with peptide 9, Table [129]IV). However, deletion of the benzene ring of phenylalanine at position 12 resulted in a considerably larger drop in potency in the resultant Ala-containing peptide (peptide 17, Table [130]IV), emphasizing the importance of van der Waals interactions of this benzene ring with its hydrophobic environment (Fig. [131]2). The peptide carrying the double replacement of V8A together with F12A (peptide 18, Table [132]IV) had lost much of its inhibitory potency and the double mutant peptide containing ∼Ala^11-Ala^12∼ (peptide 19) was virtually ineffective as an inhibitor, even at pH 4.7. The triple mutant in which all three of the front-end cluster residues were changed to ∼Ala^8-X-X-Ala^11-Ala^12∼ (peptide 20, Table [133]IV) was completely inactive as an inhibitor. View this table: * [134]In this window * [135]In a new window Table IV Interactions between yeast proteinase A and synthetic peptide forms of IA[3] [136]Figure 2 View larger version: * [137]In this window * [138]In a new window * [139]Download as PowerPoint Slide Figure 2 Stereo representation of the interactions made by the side chains of Val^8 and Phe^12 in the IA[3] polypeptide with cognate hydrophobic residues in proteinase A. Proteinase A residues are green while the Val^8 and Phe^12 side chains and the appropriate segment of the IA[3] helical backbone are inbrown. A similar response was quantified when the residues contributing to the ∼Val^26-X-X-hydrophobic-Phe^30∼ cluster at the back end of the helix were replaced. Substitution of Phe^30 by Ala, Gly, or Lys (compare peptides 21 and 22/23 with peptide 10, Table [140]IV) resulted in a significant loss in potency, again emphasizing the contribution to binding by appropriate positioning of the large benzene ring of the side chain of Phe^30. In contrast, replacement of Val^26 by Ala did not diminish inhibitory potency. Rather, it appeared to improve the binding interaction at pH 3.1 marginally (peptide 24, Table [141]IV). Replacement of the -CH[3] side chain of Ala with the -CH[2]-COOH side chain of an Asp at position 26 diminished the inhibitory potency at pH 4.7 by about 70-fold (compare peptides 25 and 24, Table [142]IV), commensurate with the introduction of a hydrophilic side chain into a hydrophobic environment. However, theK [i] value measured at pH 3.1 for peptide 25 was tighter than that derived at pH 4.7 (Table [143]IV). Of all the inhibitors listed in Tables [144]I, [145]II, and [146]IV, this was the only occasion when such an effect was observed and most likely is a reflection of the Asp side chain in its protonated and therefore uncharged form being less unfavorable in its contact with the hydrophobic environment offered by the enzyme. The double mutant peptide carrying the V26D/F30K substitutions was completely ineffective as an inhibitor (peptide 26, Table [147]IV), indicating that introduction of two hydrophilic, charged residues was highly unfavorable since there are no H-bond partners available in the enzyme to compensate for desolvation of the two side chain functions. However, amphipathic helices are often stabilized by electrostatic interactions between residues at positions i andi + 4 ([148]23) and, indeed exactly such a salt bridge is present between the Lys^24 and Asp^28 residues on the hydrophilic face of the IA[3] helix when complexed with its target proteinase (Fig. [149]1). However, the attempt to encourage Asp^26 and Lys^30 to interact with one another to form an additional salt bridge in the V26D/F30K double mutant peptide was clearly not tolerated on the hydrophobic face of the amphipathic helix in the active site cleft of the enzyme. A further mutant was also constructed in which the sequence of ∼Ser^27-Asp^28-Ala^29-Phe^30-Lys^31-Lys^32∼ was shuffled to ∼Lys^27-Ala^28-Asp^29-Lys^30-Phe^31-Ser^32∼ in the protein form of the IA3 inhibitor (Mix in Table [150]I). In this arrangement, Val^26 was retained but the salt bridge between Lys^24 and Asp^28 on the hydrophilic face of the helix was disrupted and the crucial Phe^30 residue was replaced by lysine, as in peptide 23. The resultant mutant protein (Mix, in Table [151]I) was purified to homogeneity from E. coliand found to have a K [i] value at pH 4.7 comparable to that observed for the single F30K mutant peptide (peptide 23, Table[152]IV). This might be interpreted to indicate that the salt bridge interaction between Lys^24 and Asp^28 on the hydrophilic surface of the IA[3] helix is, not unexpectedly, weak. 5 Central Residues in the Inhibitor Helix 5 The centerpiece of the inhibitory 234 residues of IA[3] is the ∼Lys^18-Leu^19-X-X-Asp^22∼ sequence. In the three crystal structures of the K24M and K31M/K32M mutant proteins and peptide 1 forms of IA3 complexed with proteinase A, the ε-NH[2] group of Lys^18 of the inhibitor hydrogen bonds to one of the carboxyl oxygens of Asp^32 of the enzyme (Fig. [153]3). This is one of the two catalytic Asp residues that operate the catalytic mechanism of all aspartic proteinases ([154]24). The ε-NH[2] group of Lys^18 also hydrogen bonds to one of the carboxyl oxygens of the side chain of Asp^22 in the IA[3] inhibitory sequence (Fig. [155]3). The other oxygen of the side chain COOH of Asp^22 hydrogen bonds to the phenolic OH group of Tyr^75 in the enzyme, a residue that is totally conserved in all eukaryotic aspartic proteinases and which is positioned almost at the tip of the β-hairpin loop or flap that overlays the active site cleft in these enzymes. A network of interactions thus cross-links these charged residues of IA[3]with the catalytically essential and structurally conserved residues of the target enzyme (Fig. [156]3). When the charged side chain of Asp^22 in the full-length protein form of IA[3] was replaced with the hydrophobic but otherwise almost isosteric side chain of a leucine residue, the purified D22L mutant protein had a slightly reduced potency both at pH 3.1 and 4.7 (Table [157]I) but nevertheless was still an effective inhibitor. [158]Figure 3 View larger version: * [159]In this window * [160]In a new window * [161]Download as PowerPoint Slide Figure 3 Stereo representation of the interactions made by the ∼Lys^18-Leu^19-X-X-Asp^22∼ centerpiece residues of IA[3] in the vicinity of the active site of proteinase A. Proteinase A residues are ingreen with the catalytic water molecule depicted as ablue sphere. The side chains of Lys^18, Leu^19, and Asp^22 plus the relevant segment of the IA[3] helix backbone are in brown. Hydrogen bonding distances are shown. However, when Lys^18 was changed to Met in concert with the D22L mutation, the resultant inhibitor (K18M/D22L, Table [162]I) was an extremely potent inhibitor. For the first time in all of these studies, it was not possible to derive an accurateK [i] value at pH 3.1 because the protein-protein interaction was so tight. This may be a reflection of the increased propensity of Met and Leu residues to be accommodated within a helical conformation by comparison with their wild-type Lys and Asp counterparts. Alternatively, this may be a further indication of the importance of hydrophobic contributions to binding strength. Modeling studies suggest that the side chain of a methionine at position 18 in the IA[3] sequence is surrounded by the hydrophobic side chains of Ile^30, Tyr^75, Thr^111, Phe^112, Phe^117, and Ile^120 of proteinase A, as well as the newly introduced side chain of Leu^22 in the K18M/D22L double mutant inhibitor. The side chain of Leu^22 can make potential, favorable interactions with C-β of Ser^35 and the side chains of Ile^73 and Tyr^75 from the flap of proteinase A, as well as with the Met^18 and Val^25 residues of the IA[3] inhibitory sequence. The importance of hydrophobic interactions was further corroborated when the Leu^19 residue within the ∼Lys^18-Leu^19-X-X-Asp^22∼ centerpiece was changed to Ala in the peptide form of IA[3]. The resultant peptide (peptide 27, Table [163]IV) was no longer an inhibitor at pH 3.1 and an apparent inhibition constant of 700 nm was estimated at pH 4.7. This loss in potency (compare peptides 27 and 9, Table [164]IV) was the largest observed for any single amino acid replacement. However, when peptide 27 was incubated with proteinase A for 16 h at pH 4.7 and 37 C at a molar ratio of 40:1, it was cleaved as a substrate (as monitored by reverse phase fast protein liquid chromatography, not shown). Peptide 27 is thus a good alternative substrate at pH 4.7 and was giving only an apparent inhibition of the activity of proteinase A toward the chromogenic substrate used in the inhibition assays. The deletion of the terminal isopropyl moiety of the leucine side chain thus converted a highly potent polypeptide inhibitor into a regular substrate of proteinase A by decreasing affinity to the target enzyme and possibly by reducing internal helix stability. Under the same conditions, peptides 14 and 8 (Table [165]II) which together span the 234 sequence of IA[3] were both cleaved by proteinase A (at the ∼Glu^10*Ile^11∼ and ∼Ala^29*Phe^30∼ bonds, respectively) as reported previously ([166]12). Peptide 7, despite having been synthesized deliberately to position a valine in place of the natural Ala^29 residue (Table [167]II) and thus make the potentially vulnerable ∼Ala^29*Phe^30∼ peptide bond resistant to attack (as in peptides 2 and 3, see earlier), was still cleaved albeit very slowly (50% in 16 h) upon incubation at pH 4.7 with proteinase A at a molar ratio of 40:1. Since cleavage at the mutated ∼Val^29Phe^30∼ bond was no longer an option, it was found by amino acid analysis that processing had taken place at the adjacent ∼Phe^30*Lys^31∼ bond (data not shown). Similarly, peptides 12 and 13 (Table [168]II) were digested after 72 h incubation with proteinase A at a molar ratio of 10:1, as were peptides 18, 19, 20, and 26 of the peptides listed in Table [169]IV. Thus, in addition to the L19A mutant (peptide 27) as described above, the other peptides (7, 8, 12, 13, 14 (Table [170]II), 18, 19, 20, and 26 (Table [171]IV)) which were not effective as inhibitors of proteinase A, served instead as substrates for the enzyme. From all of these data, it is readily apparent that the inhibitory capacity of IA[3] is located within residues 234 of the sequence and that these residues need to be present as a contiguous polypeptide to achieve inhibition. The inhibitory sequence of residues is not cleaved by the target enzyme but only very subtle alterations in sequence are necessary to convert the IA[3] polypeptide into a substrate for proteinase A. In contrast, however, the wild-type polypeptide is readily hydrolyzed as a substrate by nontarget aspartic proteinases such as pepsin and cathepsin D, which have substantial similarity to proteinase A in their primary sequences and three-dimensional structures ([172]25-27). This substantiates our NMR and CD findings reported previously ([173]12) that free IA[3] in its peptide or protein forms is predominantly unstructured. The interaction of IA[3] with its target enzyme appears to depend critically on the insertion of three hydrophobic pins (the front-end cluster, Leu^19 and the back end cluster) into the appropriate hydrophobic sockets proferred by the active site cleft of proteinase A. The target enzyme thus plays the role of a helper template, stabilizing the amphipathic helical conformation of IA[3] but, paradoxically, resulting in its own inhibition. In contrast, the random coil IA[3] polypeptide is apparently unable to locate the critical pins sufficiently precisely in its interaction with the nontarget aspartic proteinases. Consequently, since aspartic proteinases generally require only 7 or 8 amino acid residues of a polypeptide to bind in an extended β-strand conformation in their active site to act as a substrate, the nontarget enzymes are readily able to cleave IA[3]. Thus, the default setting is cleavage of the random coil IA[3]polypeptide. In contrast, helication requires many more stringent conditions to be fulfilled such as complementary amphipathicity, precision of shape, and the correct juxtaposition of side chains possessing the appropriate properties to fit snugly into the hydrophobic pockets of proteinase A. Only if IA[3] can be sculpted in situ to become the key that matches precisely into the lock that is the active site of proteinase A, can the IA[3] polypeptide escape cleavage and preserve its integrity. Thus, in its interaction with this aspartic proteinase, IA[3]is stabilized in the somewhat abnormal helical conformation, shaped by the proteinase itself but yet at the same time, held clear of the catalytic machinery through formation of the helix. The alternative, as a consequence of any imprecision of fit, is recognition in an extended conformation and cleavage. The present study has thus afforded considerable insight into the features that are important in governing the potency and selectivity of inhibition of proteinase A by IA[3]. This presents a fascinating challenge for future investigation to determine whether this unprecedented mode of inhibitor-enzyme interaction can be exploited to generate selective inhibitors re-targeted against other aspartic proteinases including those produced by pathogenic organisms. [174]Previous Section[175]Next Section 2 ACKNOWLEDGEMENTS 2 It is a pleasure to acknowledge the valuable contributions made to this project by our colleagues Anette Bruun (Carlsberg Laboratory), Alfred Chung (Protein Chemistry Core, University of Florida), Simon Cater (Cardiff School of Biosciences), and Jerry Alexandratos (NCI-Frederick). We are also very grateful to Professor Pat Sullivan and Drs. Peter Farley and William Laing, New Zealand, for generously providing the proteinase fromGlomerella cingulata; Drs Niamh Cawley and Y. Peng Loh, National Institutes of Health, Bethesda, MD, who very kindly performed the inhibition experiments with peptide 1 on yapsin 1; and Professor Simon Gaskell, Center for Mass Spectrometry, UMIST, UK, for providing ready access to mass spectrometer facilities. [176]Previous Section[177]Next Section 2 Footnotes 2 * [178]↵* This work was supported in part by a grant from the BBSRC, UK (to J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and the structure factors (code ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ ([179]http://www.rcsb.org/). * [180]↵FNb Supported by an award from Actelion, Allschwil, Switzerland. * [181]↵FNj To whom correspondence should be addressed. Tel.: 44-29-20-87-41-24; Fax: 44-29-20-87-4116; E-mail: KayJ@Cardiff.ac.uk. * Published, JBC Papers in Press, October 19, 2000, DOI 10.1074/jbc.M008520200 * Abbreviations: PCR polymerase chain reaction ACTH adrenocorticotropic hormone MALDI-TOF matrix-assisted laser desorption-time of flight MES 4-morpholineethanesulfonic acid * + Received September 18, 2000. + Revision received October 6, 2000. * The American Society for Biochemistry and Molecular Biology, Inc. [182]Previous Section 2 REFERENCES 2 1. [183]↵ 1. Vassar R., 2. Bennett B. D., 3. Babu-Khan S., 4. Kahn S., 5. Mendiaz E. A., 6. Denis P., 7. Teplow D. B., 8. Ross S., 9. Amarante P., 10. Loeloff R., 11. Luo Y., 12. Fisher S., 13. Fuller J., 14. Edenson S., 15. Lile J., 16. Jaronsinski M. A., 17. Biere A. L., 18. Curran E., 19. Burgess T., 20. Louis J-C., 21. Collins F., 22. Treanor J., 23. Rogers G., 24. Citron M. (1999) Science 286:735741. 2. [184]↵ 1. Bennett B. D., 2. Babu-Khan S., 3. Loeloff R., 4. Louis J-C., 5. 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