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Plasmodium falciparum Histo-Aspartic Protease (HAP) Protein

Kerri-Lynn Conrad '11 and Chinagozi Ugwu '10


I. Introduction

The Plasmodium falciparum Histo-aspartic protease(HAP) is one of 10 plasmepsins (PMs) identified in the genome of Plasmodium falciparum, the parasite responsible for the most widespread form of malaria. Plasmepsins are aspartic acid proteases that catalyze the hydrolysis of peptide bonds. HAP is one of 4 PMs residing in the food vacuole of the parasite (5).

Plasmodium falciparum uses host erythrocyte hemoglobin as a major nutrient source. The parasite ingests and degrades host erythrocyte hemoglobin by means of a specialized structure called a cytostome, which spans between the membranes of the erythrocyte and the parasite cytoplasm. Hemoglobin is packaged into vesicles (3), which are transported from the cytostome to the food vacuole, where it is broken down.

The active site of HAP, containing catalytic aspartate and histidine residues, has been studied as a potential target for novel antimalarial therapy (5). By inhibiting catalytic residues in the active site, the protease activity of HAP would be inhibited. Knockout of HAP's protease function could potentially preserve hemoglobin of erythrocytes infected with Plasmodium falciparum.

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II. General Structure and Active Site

HAP is a dimer composed of 2 identical monomers, A and B. Together, the 2 monomers form a large substrate binding cleft, though each monomer  can bind an individual substrate. Although HAP exists as a dimer, there is no proof, to date, that dimerization is required for enzymatic activity. The amino and carboxy terminal domains of the HAP chains are assembled into characteristic 6-stranded interdomain β-sheets that suture domains together.  

The tight interactions between the 2 monomers have a profound impact on the conformation of a functionally important loop, the "flap" residues 70-83. This "flap" remains in an open conformation in the apoenzyme. 

The active site of HAP is located in a large formed by the  amino and carboxy terminal domains of each monomer. His32 and Asp215 are the two catalytic residues located in the active site of each monomer of the protein. These residues contribute directly to HAP's protease function. 

An unusual feature of unliganded HAP is the presence of a Zn in the active site of each monomer the enzyme, as well as 4 other Zn ions on the surface of the HAP dimer.  

The Zn ion in the active site is tetrahedrally coordinated by the side chains of His32 and Asp215 from one HAP monomer, Glu278A from the other monomer, and a H2O molecule. The presence of a metal ion in the active sites of aspartic proteases has not been observed before. Although the coordination of the Zn ion in the active site of HAP resembles the active site of known metalloproteases, it is not clear whether HAP functions as a metalloprotease toward synthetic peptides. If HAP does function as a metalloprotease, it may promote nucleophilic attack on the carbonyl carbon of the protein by the oxygen atom of the aspartic acid residue, or the water molecule. 

III. Pepstatin A Binding

Pepstatin A is a potent inhibitor of aspartyl proteases. It is a hexa-peptide containing the unusual amino acid statine, having the sequence Iva-Val-Val-Sta-Ala-Sta.

Pepstatin A forms hydrogen bonds with His32 and Asp215 in the active site of HAP. Pepstatin A has 2 hydroxyl groups on side chain residues. They are located 2 residues from one another. The residue closer to the amino end of Pepstatin A participates in a hydrogen bond with His32 , while the residue closer to the carboxy end of Pepstatin A participates in a hydrogen bond with Asp215 . Because the nucleophilic O and N atoms of Asp215 and His32, respectively, are stabalized by strong hydrogen bonds with Pepstatin A hydroxyl residues, Asp215 and His32 can no longer participate in catalysis of peptide bonds via nucleophilic attack at carbonyl residues of peptide bonds.

When Pepstatin A binds with HAP in the active site, the "flap" located at residues 70-83 shifts to a "closed" conformation . The change in conformation is most dramatic in  the Lys76 residue, which shifts 6.9 angstroms from the open position in the apoenzyme. The Lys76 residue participates in a hydrogen bond with the carbonyl oxygen at the carboxy terminus of an alanine residue of the inhibitor . This is the only bond the inhibitor forms to the flap, due to the orientation of the carboxy terminus when the flap "closes" upon Pepstatin A binding.

Because Pepstatin A cannot form bonds to the conformationally "closed" flap upon binding to HAP, the primary means of inhibition of HAP protease function is by hydrogen bonding to the catalytic His32 and Asp215 residues in the active site.

IV. KNI-10006 Binding

KNI-10006 is a peptidomimetic inhibitor, a family of inhibitors aimed as anti-HIV agents targeting the retroviral protease. KNI-10006 is a potent inhibitor of HAP, with an IC50=0.69 µM

The binding mode of KNI-10006 to HAP is drastically different than that of Pepstatin A. In the KNI-10006-HAP complex, the hydroxyl group at the central part of the inhibitor points away from the catalytic His32 and Asp215 residues in the active site.

The flap is in an "open" conformation in the KNI-10006-HAP complex, creating a "flap pocket" with an abundance of hydrophobic residues. KNI-10006 interacts with the hydrophobic residues of flap 70-83, including Val71, Leu73, Thr74,Ala77, Gly78, Thr79, Ile80, and Gly82.

The Phe111 residue at the entrance of the flap pocket is unique to HAP. KN1-10006 displays strong hydrophobic interactions with this residue . It is believed that Phe111 substitution in HAP as opposed to other plasmepsin proteases such as PMII and PMIV plays a role in HAP's ligand preference.

V. Implications for Anti-Malarial Drug Design

Pepstatin A and KNI-10006 have been identified as potent inhibitory drugs to target the P. falciparum HAP proteins. These compounds are particularly exciting for the development of anti-malarial drugs because they are adaptive inhibitors of the P. falciparum aspartyl protease family (4).

The Pepstatin A peptide greatly reduces the rate of erythrocyte degradation by inhibiting the activity of the P. falciparum digestive vacuole. Pepstatin A particularly reduces the activity of the four most lethal P. falciparum aspartic proteases (plasmepsins) I, II, IV and HAP (6).  This peptide binds to the active site of the HAP enzyme and blocks P. falciparum catalytic protein from degrading the hemoglobin cells.

KNI-10006 does not bind to the active site of the HAP protein unlike most other anti-malarial drug. This compound inhibits the four most virulent malarial plasmepsins at nanomolar concentrations (3,6). However the potency with which KNI-10006 peptide inhibits the parasitic plasmepsins II and IV is much greater than all the other parasitic plasmepsins.

The inhibitory concentration 50 (IC50) values for plasmepsins II and IV with the KNI-10006 compound is 39 nM and 15 nM respectively (6). The KNI-10006 poses a problem in drug design in that it inhibits human aspartic protease. Fortunately KNI-10006 only inhibits human aspartyl protease pepsin at several orders of magnitude less that it inhibits P. falciparum (3,5).

As a consequence of KNI-10006 cell/vacuole membrane impermeability, the capacity to which parasite growth in infected erythrocyte cultures with P. falciparum is extremely low (4,6). Experimentation is ongoing to develop a more permeable KNI-10006.

VI. References

  1. Bhaumik, P., Xiao, H., Parr, C. L., Kiso, Y., A, Gustchina., Yada, R. Y., A. Wlodawer. 2009.Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum. Mol. Biol. 388:520–540.


  1. Hidaka, K., Kimura, T., Tsuchiya, Y., Kamiya, M., Ruben, A. J., Freire, E., Hayashia, Y., Kisoa, Y. 2007. Additional interaction of allophenylnorstatine-containing tripeptidomimetics with malarial aspartic protease plasmepsin II. Bioorganic & Medicinal Chemistry Letters. 17:3048–3052.


  1. Kiso, A., Hidaka, K., Kimura, T., Hayashi, Y., Nezami, A., Ernesto, F., Kiso, Y. 2004. Search for Substrate-Based Inhibitors Fitting the S2’ Space of Malarial Aspartic Protease Plasmepsin II. J. Peptide Sci.10:641-647.


  1. Nezami, A., Kimura, T., Hidaka, K., Kiso, A., Liu, J., Kiso, Y. Goldberg, D. E., Freire, E. 2003. High-Affinity Inhibition of a Family of Plasmodium falciparum Proteases by a Designed Adaptive Inhibitor. Biochemistry. 42: 8459-8464.


  1. Nguyen, J.T., Hamada, Y., Kimura, T., Kiso, Y. 2008. Design of Potent Aspartic Protease Inhibitors to Treat Various Diseases. Arch. Pharm. Chem. Life Sci. 341: 523 – 535.


  1. Orrling, K. M., Marzahn, M. R., Gutiérrez-de-Terán, H., Åqvist, J., Dunn, B. M., Larhed, M. 2009. α-Substituted norstatines as the transition-state mimic in inhibitors of multiple digestive  vacuole malaria aspartic proteases. Bioorganic & Medicinal Chemistry. 17:5933–5949.


  1. Silva, A.M., Lee, A.Y., Gulnik, S.V., Majer, P.,  Collins, J., Bhat, T.N., Collins, P.J., Cachau, R.E., Luker, K.E., Gluzman, I.Y., Francis, S.E., Oksman, A., Goldberg, D.E., Erickson, J.W. 1996. Structure and Inhibition of Plasmepsin II, a Hemoglobin-Degrading Enzyme from Plasmodium falciparum. Proc. Natl. Acad. Sci. 93: 10034-10039.


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