Huogen Xiao

The zymogen of plasmepsin V from Plasmodium falciparum is enzymatically active

Plasmepsin V, a membrane-bound aspartic protease present in Plasmodium falciparum, is involved in the export of malaria parasite effector proteins into host erythrocytes and therefore is a potential target for antimalarial drug development. The present study reports the bacterial recombinant expression and initial characterization of zymogenic and mature plasmepsin V. A 484-residue truncated form of proplasmepsin (Glu37-Asn521) was fused to a fragment of thioredoxin and expressed as inclusion bodies. Refolding conditions were optimized and zymogen was processed into a mature form via cleavage at the Asn80-Ala81 peptide bond. Mature plasmepsin V exhibited a pH optimum of 5.5-7.0 with Km and kcat of 4.6 μM and 0.24s(-1), respectively, at pH 6.0 using the substrate DABCYL-LNKRLLHETQ-E(EDANS). Furthermore, the prosegment of proplasmepsin V was shown to be nonessential for refolding and inhibition. Unexpectedly, unprocessed proplasmepsin V was enzymatically active with slightly reduced substrate affinity (∼ 2-fold), and similar pH optimum as well as turnover compared to the mature form. Both zymogenic and mature plasmepsin V were partially inhibited by pepstatin A as well as several KNI aspartic protease inhibitors while certain metals strongly inhibited activity. Overall, the present study provides the first report on the nonessentiality of the prosegment for plasmepsin V folding and activity, and therefore, subsequent characterization of its structure-function relationships of both zymogen and mature forms in the development of novel inhibitors with potential antimalarial activities is warranted.

The catalytic significance of the proposed active site residues in Plasmodium falciparum histoaspartic protease

Alanine mutations of the proposed catalytically essential residues in histoaspartic protease (HAP) (H34A, S37A and D214A) were generated to investigate whether: (a) HAP is a serine protease with a catalytic triad of His34, Ser37 and Asp214 [Andreeva N, Bogdanovich P, Kashparov I, Popov M & Stengach M (2004) Proteins55, 705–710]; or (b) HAP is a novel protease with Asp214 acting as both the acid and the base during substrate catalysis with His34 providing critical stabilization [Bjelic S & Aqvist J (2004) Biochemistry43, 14521–14528]. Our results indicated that recombinant wild‐type HAP, S37A and H34A were capable of autoactivation, whereas D214A was not. The inability of D214A to autoactivate highlighted the importance of Asp214 for catalysis. H34A and S37A mutants hydrolyzed synthetic substrate indicating that neither His34 nor Ser37 was essential for substrate catalysis. Both mutants did, however, have reduced catalytic efficiency (P ≤ 0.05) compared with wild‐type HAP, which was attributed to the stabilizing role of His34 and Ser37 during catalysis. The mature forms of wild‐type HAP, H34A and S37A all exhibited high activity over a broad pH range of 5.0–8.5 with maximum activity occurring between pH 7.5 and 8.0. Inhibition studies indicated that wild‐type HAP, H34A and S37A were strongly inhibited by the serine protease inhibitor phenylmethanesulfonyl fluoride, but only weakly inhibited by pepstatin A. The data, in concert with molecular modeling, suggest a novel mode of catalysis with a single aspartic acid residue performing both the acid and base roles.

The native conformation of plasmepsin II is kinetically trapped at neutral pH

Plasmepsin II (PMII), an aspartic protease from the malarial parasite Plasmodium falciparum, represents a model for understanding protease structure/function relationships due to its unique structure and properties. The present study undertook a thermodynamic and kinetic analysis of the PMII folding mechanism and a pH stability profile. Differential scanning calorimetry revealed that the native state of PMII (Np) was irreversibly unfolded, and in the pH range of 6.5-8.0, PMII refolds to a denatured state (Rp) with higher thermal stability than Np. Rp could also be formed upon partially unfolding PMII at pH 11.0 and 37 °C for 2h, followed by adjustment to a pH in the range of 6.5-8.0. While Rp could be folded/unfolded reversibly, Np was shown to exist as a kinetically trapped state. By examining the unfolding kinetics of Np and the kinetics of Rp folding to Np at 25 °C, it was found that Np is kinetically trapped by an unfolding barrier of 25.5 kcal/mol, and yet once unfolded, is prevented from folding by a comparable folding barrier. The folding mechanism of PMII is similar to that reported for pepsin. It is hypothesized that the PMII zymogen also utilizes a prosegment-catalyzed folding mechanism.

Understanding the structural basis of substrate recognition by Plasmodium falciparum plasmepsin V to aid in the design of potent inhibitors

Plasmodium falciparum plasmepsin V (PfPMV) is an essential aspartic protease required for parasite survival, thus, considered as a potential drug target. This study reports the first detailed structural analysis and molecular dynamics simulation of PfPMV as an apoenzyme and its complexes with the substrate PEXEL as well as with the inhibitor saquinavir. The presence of pro-peptide in PfPMV may not structurally hinder the formation of a functionally competent catalytic active site. The structure of PfPMV-PEXEL complex shows that the unique positions of Glu179 and Gln222 are responsible for providing the specificity of PEXEL substrate with arginine at P3 position. The structural analysis also reveals that the S4 binding pocket in PfPMV is occupied by Ile94, Ala98, Phe370 and Tyr472, and therefore, does not allow binding of pepstatin, a potent inhibitor of most pepsin-like aspartic proteases. Among the screened inhibitors, the HIV-1 protease inhibitors and KNI compounds have higher binding affinities for PfPMV with saquinavir having the highest value. The presence of a flexible group at P2 and a bulky hydrophobic group at P3 position of the inhibitor is preferred in the PfPMV substrate binding pocket. Results from the present study will aid in the design of potent inhibitors of PMV.

Characterization of the monomer-dimer equilibrium of recombinant histo-aspartic protease from Plasmodium falciparum.

Histo-aspartic protease (HAP) from Plasmodium falciparum is an intriguing aspartic protease due to its unique structure. Our previous study reported the first recombinant expression of soluble HAP, in its truncated form (lys77p-Leu328) (p denotes prosegment), as a thioredoxin (Trx) fusion protein Trx-tHAP. The present study found that the recombinant Trx-tHAP fusion protein aggregated during purification which could be prevented through the addition of 0.2% CHAPS. Trx-tHAP fusion protein was processed into a mature form of tHAP (mtHAP) by both autoactivation, and activation with either enterokinase or plasmepsin II. Using gel filtration chromatography as well as sedimentation velocity and equilibrium ultracentrifugation, it was shown that the recombinant mtHAP exists in a dynamic monomer-dimer equilibrium with an increasing dissociation constant in the presence of CHAPS. Enzymatic activity data indicated that HAP was most active as a monomer. The dominant monomeric form showed a K(m) of 2.0 microM and a turnover number, k(cat), of 0.036s(-1) using the internally quenched fluorescent synthetic peptide substrate EDANS-CO-CH(2)-CH(2)-CO-Ala-Leu-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-Dap-(DABCYL)-OH (2837b) at pH 5.2.

The prosegment catalyzes native folding of Plasmodium falciparum plasmepsin II

Plasmepsin II is a malarial pepsin-like aspartic protease produced as a zymogen containing an N-terminal prosegment domain that is removed during activation. Despite structural similarities between active plasmepsin II and pepsin, their prosegments adopt different conformations in the respective zymogens. In contrast to pepsinogen, the proplasmepsin II prosegment is 80 residues longer, contains a transmembrane region and is non-essential for recombinant expression in an active form, thus calling into question the prosegments precise function. The present study examines the role of the prosegment in the folding mechanism of plasmepsin II. Both a shorter (residues 77-124) and a longer (residues 65-124) prosegment catalyze plasmepsin II folding at rates more than four orders of magnitude faster compared to folding without prosegment. Native plasmepsin II is kinetically trapped and requires the prosegment both to catalyze folding and to shift the folding equilibrium towards the native conformation. Thus, despite low sequence identity and distinct zymogen conformations, the folding landscapes of plasmepsin II and pepsin, both with and without prosegment, are qualitatively identical. These results imply a conserved and unusual feature of the pepsin-like protease topology that necessitates prosegment-assisted folding.

Deciphering the mechanism of potent peptidomimetic inhibitors targeting Plasmepsins: Biochemical and structural insights

Malaria is a deadly disease killing worldwide hundreds of thousands people each year and the responsible parasite has acquired resistance to the available drug combinations. The four vacuolar plasmepsins (PMs) in Plasmodium falciparum involved in hemoglobin (Hb) catabolism represent promising targets to combat drug resistance. High antimalarial activities can be achieved by developing a single drug that would simultaneously target all the vacuolar PMs. We have demonstrated for the first time the use of soluble recombinant plasmepsin II (PMII) for structure‐guided drug discovery with KNI inhibitors. Compounds used in this study (KNI‐10742, 10743, 10395, 10333, and 10343) exhibit nanomolar inhibition against PMII and are also effective in blocking the activities of PMI and PMIV with the low nanomolar Ki values. The high‐resolution crystal structures of PMII–KNI inhibitor complexes reveal interesting features modulating their differential potency. Important individual characteristics of the inhibitors and their importance for potency have been established. The alkylamino analog, KNI‐10743, shows intrinsic flexibility at the P2 position that potentiates its interactions with Asp132, Leu133, and Ser134. The phenylacetyl tripeptides, KNI‐10333 and KNI‐10343, accommodate different ρ‐substituents at the P3 phenylacetyl ring that determine the orientation of the ring, thus creating novel hydrogen‐bonding contacts. KNI‐10743 and KNI‐10333 possess significant antimalarial activity, block Hb degradation inside the food vacuole, and show no cytotoxicity on human cells; thus, they can be considered as promising candidates for further optimization. Based on our structural data, novel KNI derivatives with improved antimalarial activity could be designed for potential clinical use.

Structure-based drug designing against plasmepsins from Plasmodium falciparum

Vandana Mishra1, Ishan Rathore1, Anagha Arekar1, Huogen Xiao2, Rickey Yada3, Alexander Wlodawer4, Alla Gustchina4, Koushi Hidaka5, Prasenjit Bhaumik1 1Biosciences & Bioengineering. Iit Bombay, Mumbai, India, 2Department of Molecular and Cellular Biology, University of Guelph, N1G2W1., Ontario, Canada, 3Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall Vancouver, BC V6T 1Z4., Vancouver, Canada, 4Protein Structure Section, Macromolecular Crystallography laboratory, National Cancer Institute., Frederick, United States, 5Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, 1-1-3 Minatojima, Chuo-ku, Kobe 650-8586, Japan, Chuo-ku, Japan E-mail: vandana.mishra021290@gmail.com

Structural insights into the mechanism of plasmepsins from P. falciparum

Ishan Rathore1, Vandana Mishra1, Chandan Patel2, Huogen Xiao3, Rickey Yada4, Alla Gustchina5, Alexander Wlodawer5, Koushi Hidaka6, Prasenjit Bhaumik1 1Department Of Biosciences And Bioengineering, IIT-Bombay, Mumbai, India, 2Department of Chemistry, IIT-Bombay, Mumbai, India, 3Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada, 4Faculty of Land and Food Systems, University of British Columbia, Vancouver, Canada, 5Protein Structure Section, Macromolecular Crystallography Laboratory, National Cancer Institute, Frederick, United States, 6Division of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Kobe, Japan E-mail: ishanrathore21@gmail.com

Crystal structure of Histo-aspartic protease from Plasmodium falciparum

4 is a key player in the formation of large complexes on gene promoters leading to chromatin remodelling and gene activation. CARM1 has now been shown to methylate a large variety of proteins which are all vital to gene expression. CARM1 contains 608 amino acids in human and its architecture has been schematically divided into three domains. CARM1 is built around a catalytic core domain that is well conserved in sequence (and therefore in structure) among all PRMTs members. CARM1 possesses two unique additional domains attached, respectively, at the NH2-terminal and at the COOH-terminal end of the PRMT active site. Both NH2-terminal domain and COOHterminal domain have been shown to be required for the coactivator function of human CARM1. We have solved six crystal structures corresponding to three isolated modules of CARM1. Five crystal structures of the CARM1 catalytic module, two free, two cofactor and one inhibitor bound forms have revealed large structural modifications and shown that the NH2-terminal and the COOHterminal end of CARM1 catalytic module contain molecular switches that may inspire how CARM1 regulates its biological activities by protein-protein interactions. Full detailed analysis of the structures will be presented.