Characterization and structural studies of the Plasmodium falciparum ubiquitin and Nedd8 hydrolase UCHL3

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.

FIGURE 1.

Both mammalian and P. falciparum-derived Nedd8 are substrates for PfUCHL3. A, sequences of the P. falciparum and human Nedd8 orthologs were aligned to show the 52% identity between the two proteins. Residues mutated to glutamine to achieve solubility in protein expression experiments are marked with an asterisk. B, the four side chains mutated to glutamine are shown in gray on a ribbon model of PfNedd8. C, to show that PfUCHL3 is specific for PfNedd8, the parasite UBL (with the four solubilizing mutations) was N-terminally FLAG-tagged and modified with a VME on its C terminus to generate an active electrophilic probe. PfUCHL3 was expressed using in vitro coupled transcription/translation in the presence of [³⁵S]methionine and subsequently reacted with FLAG-PfNedd8-VME, mammalian Nedd8-vinylsulfone (MmNedd8-VS) or hemagglutinin-tagged ubiquitin vinyl methyl ester (HAUb-VME) in the presence or absence of 10 mm NEM to show cysteine-specific activity and analyzed by SDS-PAGE followed by autoradiography. A shift in molecular weight in the absence of NEM demonstrates reactivity. D and E, assays based on enzymatic cleavage of Ub-AMC and Nedd8-AMC show that PfUCHL3 substrate recognition happens at physiologically relevant rates when compared with HsUCHL3 and NedP1. Each enzyme was mixed with an excess of Ub-AMC (D) or Nedd8-AMC (E) substrate. Cleavage was measured by fluorescence output every 2 min for a total of 40 min.

FIGURE 2.

HA-PfUCHL3wt-expressing parasites are viable and express functional enzyme. Transgenic, HA-PfUCHL3-expressing parasites were tested by Southern blot to verify transgene presence (A). Genomic DNA from 3D7 wild-type and PfUCHL3 transgenic parasites digested with AccI/PacI and hybridized with a ³²P-labeled probe representing the PfUCHL3 sequence. The presence of the episome containing the exogenous PfUCHL3 open reading frame shown by a hybridization signal of 6033 bp. The stronger signal from the episome signifies multiple copies of the overexpression plasmid. B, crude parasite lysates derived from the untransfected parent line (WT) and the transgenic line (PfUCHL3) were reacted with Ub-VME and blotted with anti-HA.

FIGURE 3.

Sequence alignments of P. falciparum and human UCHL3, and PfNedd8 with Ub. A, the aligned PfUCHL3 and HsUCHL3 amino acid sequences. The parasitic enzyme sequence was previously determined (10). Catalytic triad residues are marked by asterisks, and the sequence corresponding to the crossover loop is boxed. B, the PfNedd8 sequence was aligned with that of human Ub. P. falciparum Ub and human Ub (HsUb) differ at a single amino acid (residue 16 is Glu or Asp in human or P. falciparum, respectively). Reagents based on the HsUb were used throughout these experiments. In both alignments, black boxes indicate identical residues, and white boxes are conservative substitutions.

FIGURE 4.

Crystal structures of free PfUCHL3 and the PfUCHL3-UbVME complex. A, ribbon representation of the PfUCHL3 structure (gray) in complex with UbVME (green). Side chains of the catalytic triad residues are shown in yellow. The dashed line represents a disordered region spanning residues 58–78. B, superimposition of the UbVME complex of HsUCHL3 (orange) and PfUCHL3 (gray). Only one bound UbVME (green), that of PfUCHL3, is shown for clarity. The disordered region of PfUCHL3 corresponds to Helix H3 in HsUCHL3. The crossover loop of both structures adopts notably different conformations. In the crystal structure of free HsUCHL3, this loop and Helix 6′ were unstructured (15). C, structure of free PfUCHL3 (blue) superimposed onto UbVME-bound PfUCHL3 (gray). The loop regions implicated in Ub recognition are indicated by arrows and undergo significant conformational changes upon Ub binding.

FIGURE 5.

Interface between the Ub C terminus and the active site of PfUCHL3. A, interactions of the extended C terminus of Ub (green) with PfUCHL3 residues (gray) lining the narrow groove. The catalytic C92 forms a covalent bond to the VME moiety replacing the C-terminal Gly-76 of Ub. Note that all backbone carbonyl and amide groups are coordinated by hydrogen bonds (dashed lines) to PfUCHL3 residues. Corresponding HsUCHL3 residues that are not conserved in PfUCHL3 are shown in orange. Hydrogen bonds only observed in the PfUCHL3-UbVME complex are shown as dashed green lines. B, 2F_o − F_c electron density map contoured at 1.3 σ indicates a covalent bond between the catalytic cysteine C92 and the former VME moiety at the Ub C terminus.

FIGURE 6.

MD simulations of PfUCHL3-PfNedd8. A, r.m.s.d. values for PfUCHL3 in complex with Nedd8. r.m.s.d. of Cα atoms were computed throughout a 10-ns simulation for the complex (green curve) and components Nedd8 (red curve) and PfUCHL3 (black curve). r.m.s.d. for modeled Nedd8 reached stable values below 1 Å after 2 ns of simulation. B, model of the PfUCHL3 active site bound to PfNedd8VME (magenta) after 10 ns of equilibration in MD simulations. The corresponding Ub C-terminal residues (green) as observed in the PfUCHL3 complex after being subjected to a similar simulation are superimposed. The conformation of Ub residue Arg-74 changes significantly during the simulation (compare with Fig. 4A) and resembles that of Nedd8 Arg-74, forming a salt bridge to Asp-157. Simulations suggest that Nedd8 Gln-72 interacts with Glu-11 and Asn-13 of PfUCHL3.