Lowri H. Phylip

High level expression and characterisation of Plasmepsin II, an aspartic proteinase from Plasmodium falciparum

DNA encoding the last 48 residues of the propart and the whole mature sequence of Plasmepsin II was inserted into the T7 dependent vector pET 3a for expression in E. coli. The resultant product was insoluble but accumulated at ∼20 mg/l of cell culture. Following solubilisation with urea, the zymogen was refolded and, after purification by ion‐exchange chromatography, was autoactivated to generate mature Plasmepsin II. The ability of this enzyme to hydrolyse several chromogenic peptide substrates was examined; despite an overall identity of ∼35% to human renin, Plasmepsin II was not inhibited significantly by renin inhibitors.

The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix.

Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae . Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 Å, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2–34, reveal an unprecedented mode of inhibitor–enzyme interactions. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2–32 become ordered and adopt a near-perfect α-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases.

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.

Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1.

A series of synthetic, chromogenic substrates for HIV‐1 proteinase with the general structure Ala‐Thr‐His‐Xaa‐Yaa‐Zaa∗Nph‐Val‐Arg‐Lys‐Ala was synthesised with a variety of residues introduced into the Xaa, Yaa and Zaa positions. Kinetics parameters for hydrolysis of each peptide by HIV‐1 proteinase at pH 4.7, 37°C and u = 1.0 M were measured spectrophotometrically and/or by reverse phase FPLC. A variety of residues was found to be acceptable in the P3, position whilst hydrophobic/aromatic residues were preferable in P1. The nature of the residue occupying the P2; position had a strong influence on k cat (with little effect on k m;β‐branched residues Val or Ile in this position resulted in considerably faster peptide hydrolysis than when e.g. the Leu‐containing analogue was present in P2.

Escape mutants of HIV-1 proteinase: enzymic efficiency and susceptibility to inhibition

Genes encoding a number of mutants of HIV-1 proteinase were sub-cloned and expressed in E. coli. The proteinases containing mutations of single residues (e.g., G48V, V82F, I84V and L90M) were purified and their catalytic efficiencies relative to that of wild-type proteinase were examined using a polyprotein (recombinant HIV-1 gag) substrate and several series of synthetic peptides based on the -Hydrophobic * Hydrophobic-, -Aromatic * Pro- and pseudo-symmetrical types of cleavage junction. The L90M proteinase showed only small changes, whereas the activity of the other mutant enzymes was compromised more severely, particularly towards substrates of the -Aromatic * Pro- and pseudo-symmetrical types. The susceptibility of the mutants and the wild-type proteinase to inhibition by eleven different compounds was compared. The L90M proteinase again showed only marginal changes in its susceptibility to all except one of the inhibitors examined. The K(i) values determined for one inhibitor (Ro31-8959) showed that its potency towards the V82F, L90M, I84V and G48V mutant proteinases respectively was 2-, 3-, 17- and 27-fold less than against the wild-type proteinase. Several of the other inhibitors examined form a systematic series with Ro31-8959. The inhibition constants derived with these and a number of other inhibitors, including ABT-538 and L-735,524, are used in conjunction with the data on enzymic efficiency to assess whether each mutation in the proteinase confers an advantage for viral replication in the presence of any given inhibitor.

Bacterial aspartic proteinases

Regions of genomic DNA encoding putative aspartic proteinase domains were amplified by PCR from the bacterial species, Escherichia coli and Haemophilus influenzae. Expression of each of these DNA fragments resulted in the accumulation of the corresponding recombinant proteins in insoluble aggregates. Each recombinant protein was solubilised, refolded and shown to be able to cleave synthetic peptides that have been extensively used previously as substrates for aspartic proteinases of vertebrate, fungal and retroviral origin. Each activity was completely blocked by the diagnostic aspartic proteinase inhibitor, acetyl‐pepstatin. This is thus the first report demonstrating unequivocally that aspartic proteinases may be present in bacteria.

Aspartic proteinase inhibitors from tomato and potato are more potent against yeast proteinase A than cathepsin D

The interaction of a variety of aspartic proteinases with a recombinant tomato protein produced in Pichia pastoris was investigated. Only human cathepsin D and, even more potently, proteinase A from Saccharomyces cerevisiae were inhibited. The tomato polypeptide has >80% sequence identity to a previously reported potato inhibitor of cathepsin D. Re-evaluation of the potato inhibitor revealed that it too was more potent (>20-fold) towards yeast proteinase A than cathepsin D and so might be renamed the potato inhibitor of proteinase A. The potency towards yeast proteinase A may reflect a similarity between this fungal enzyme and aspartic proteinases produced by fungal pathogens which attack tomato and/or potatoes.

Intrinsic activity of precursor forms of HIV-1 proteinase

The wild‐type ‐Phe*Pro‐ bond located at the N‐terminus of the mature aspartic proteinase of HIV‐1 was replaced by ‐Ile‐Pro‐ or ‐Val‐Pro‐. By this means, processing at this cleavage junction was prevented and so, extended or precursor forms of HIV‐proteinase were generated. These constructs were expressed in Escherichia coli, purified therefrom, and their specificity, activity at different pH values and susceptibility to the potent inhibitor, Ro31‐8959, was assessed. A hitherto unobserved cleavage junction (at ∼Ala‐Phe*Leu‐Gln∼) in the frame‐shift region of the gag‐pol viral penome was identified and confirmed by demonstrating cleavage of a synthetic peptide corresponding to this region. The implications for viral replication of self‐processing at neutral pH by proteinase whilst still present (in a precursor form) as a component of the polyprotein are considered; such reactions, however, are still blocked even at pH values as high as 8.0 by Ro31‐8959.

MUTATING P2 AND P1 RESIDUES AT CLEAVAGE JUNCTIONS IN THE HIV-1 POL POLYPROTEIN : EFFECTS ON HYDROLYSIS BY HIV-1 PROTEINASE

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (‐Leu*Phe‐) and (b) between the p51 and RNase H domain (‐Phe*Tyr‐) within p66 of RT in the HIV‐1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a β‐branched (He) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a β‐branched (Val) residue in the P1 position flanking such ‐Hydrophobic*Hydrophobic‐ junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.

Key Features Determining the Specificity of Aspartic Proteinase Inhibition by the Helix-forming IA3 Polypeptide

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.