Characterization and Structural Studies of the Plasmodium falciparum Ubiquitin and Nedd8 Hydrolase UCHL3

Bradley I Coleman,Hidde L Ploegh,Rachelle Gaudet,Christy A Comeaux,Wilhelm A Weihofen,Katerina Artavanis-Tsakonas,John M Antos,Manoj T Duraisingh

doi:10.1074/jbc.m109.072405

Bradley I Coleman, Hidde L Ploegh + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m109.072405

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Abstract

Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed the identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
We previously identified PfUCHL3 as a functional deubiquitinating enzymes (DUBs) in P. falciparum
Instead of limiting the size of substrates, the loop can rotate to allow catalysis of larger targets, larger proteins have been found to be inefficiently deubiquitinated by UCHL3 [19]

Summary

EXPERIMENTAL PROCEDURES

Generation of Electrophilic Probes—Probes were generated as described in a previous study [8]. Maintenance of Parasite Cultures—The 3D7 strain of P. falciparum was obtained from the WEHI (Melbourne, Australia), and the 3D7-attB strain was obtained from the Fidock laboratory at Columbia University (New York) Both were maintained in in vitro culture as previously described [23]. Mixed stage parasites were lysed using 1% Nonidet P-40 in the presence of protease inhibitors (Roche Applied Science) and lysate (40 ␮g of protein) was incubated for 1 h at room temperature in the presence or absence of Ub-VME and subsequently separated by SDS-PAGE. The crystal structure of PfUCHL3-UbVME was determined by molecular replacement with PHASER [28] using the components of the HsUCHL3-UbVME structure as search models (Protein Data Bank code 1xd). Overall structural changes of the proteins were monitored with VMD by computing r.m.s.d. values of C␣ atoms over the entire trajectories, using the crystallographic structure (or starting homology model in the case of the PfNedd complex) as a reference point

RESULTS

Observed reflections

DISCUSSION