The Crystal Structure of Cruzain: A Therapeutic Target for Chagas' Disease

McGrath, Mary E.; Eakin, Ann E.; Engel, Juan C.; McKerrow, James H.; Craik, Charles S.; Fletterick, Robert J.

doi:10.1006/jmbi.1994.0137

Cited by 246 publications

(201 citation statements)

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“…Interestingly, this procedure identified cruzipain (also known as cruzain), the major cysteine protease of the blood parasite T. cruzi (the causative agent of Chagas disease) (45)(46)(47), as the most similar structural relative to falcipain-2, with a backbone root mean square deviation (r.m.s.d.) of 1.7 Å over 207 homologous residues.…”

Section: Refolding and Purification Of The Falcipain-2 Mutant Forms-mentioning

confidence: 99%

Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

Hogg

Nagarajan

Herzberg

et al. 2006

Journal of Biological Chemistry

111

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Malaria is caused by protozoan erythrocytic parasites of the Plasmodium genus, with Plasmodium falciparum being the most dangerous and widespread disease-causing species. Falcipain-2 (FP-2) of P. falciparum is a papain-family (C1A) cysteine protease that plays an important role in the parasite life cycle by degrading erythrocyte proteins, most notably hemoglobin. Inhibition of FP-2 and its paralogues prevents parasite maturation, suggesting these proteins may be valuable targets for the design of novel antimalarial drugs, but lack of structural knowledge has impeded progress toward the rational discovery of potent, selective, and efficacious inhibitors. As a first step toward this goal, we present here the crystal structure of mature FP-2 at 3.1 Å resolution, revealing novel structural features of the FP-2 subfamily proteases including a dynamic ␤-hairpin hemoglobin binding motif, a flexible N-terminal ␣-helical extension, and a unique active-site cleft. We also demonstrate by biochemical methods that mature FP-2 can proteolytically process its own precursor in trans at neutral to weakly alkaline pH, that the binding of hemoglobin to FP-2 is strictly pH-dependent, and that FP-2 preferentially binds methemoglobin over hemoglobin. Because the specificity and proteolytic activity of FP-2 toward its multiple targets appears to be pH-dependent, we suggest that environmental pH may play an important role in orchestrating FP-2 function over the different life stages of the parasite. Moreover, it appears that selectivity of FP-2 for methemoglobin may represent an evolutionary adaptation to oxidative stress conditions within the host cell.

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Section: Refolding and Purification Of The Falcipain-2 Mutant Forms-mentioning

confidence: 99%

Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

Hogg

Nagarajan

Herzberg

et al. 2006

Journal of Biological Chemistry

111

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“…The papain [7], actinidin [8], human liver cathepsin B [9], caricain D158E mutant complexed with E-64 was concentrated to approx. 13 [10], cruzain [11] and glycyl endopeptidase [12]. There are also mg/ml in 10 mM Tris pH 8.0.…”

Section: Preparation and Crystallizationmentioning

confidence: 99%

Crystal structure of a caricain D158E mutant in complex with E‐64

et al. 1996

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'similar' hydrogen bonds to the Asp but the Glu could not. Abstract The structure of the D158E mutant of caricain (previously known as papaya protease omega) in complex withThese authors clearly consider that Asn could fit neatly into E-64 has been determined at 2.0 A resolution (overall R factor the unperturbed structure whilst the Glu side chain is too long 19.3%). The structure reveals that the substituted glutamate to fit. Taylor et al. [19] report the effect of substituting Ala, makes the same pattern of hydrogen bonds as the aspartate in Asn and Glu for Asp-158 in caricain and highlight the potennative caricain. This was not anticipated since in the native tial multiple ionizations involved in activity. The kcJKm valstructure there is insufficient room to accommodate the ues for the caricain mutants rank Asp > Glu >Asn > Ala. glutamate side chain. The glutamate is accommodated in the This suggests that for caricain the Glu-158 mutant is the mutant by a local expansion of the structure demonstrating that most similar in structure as it has the highest activity. Clearly, small structural changes are responsible for the change in the same hydrogen bonds could be made by the glutamate if it activity, could be accommodated within the structure.To establish the position of the glutamate side chain at Key words: Kinetically influential mutant; Cysteine protease; Site directed mutagenesis; X-ray crystallography; position 158 in caricain we have determined the structure by Enzyme-inhibitor complex X-ray crystallography. We discuss this structure and activity, and the implications for understanding the effect of mutations at the corresponding position in papain.

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“…Inhibition of cruzain activity with fluoromethyl ketone-based inhibitors seems to be correlated with the loss of feasibility of parasites in both ex-vivo tissue culture and in vivo mouse models. 19,32,33 Information derived from the study of the molecular mechanism of hydrolysis catalyzed by cysteine proteases could be used as starting point to explore the inhibition mechanism at atomistic level. Early studies revealed a participation of the residues Cys25 and His159 (cruzain numbering) from the active site of theses proteases.…”

Section: Introductionmentioning

confidence: 99%

First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones

2015

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Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics−molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen−carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.

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The Crystal Structure of Cruzain: A Therapeutic Target for Chagas' Disease

Cited by 246 publications

References 0 publications

Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

Structural and Functional Characterization of Falcipain-2, a Hemoglobinase from the Malarial Parasite Plasmodium falciparum

Crystal structure of a caricain D158E mutant in complex with E‐64

First Quantum Mechanics/Molecular Mechanics Studies of the Inhibition Mechanism of Cruzain by Peptidyl Halomethyl Ketones

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