Structural basis of LHVS inhibition. A, surface representation of the TgCPL active site cleft with modeled LHVS shown as balls and sticks. The catalytic triad is colored magenta, and the view is the same as that depicted in Fig. 4, B and C. B, stereoview of the LHVS binding mode. The surface of TgCPL has been removed, and amino acids within 5 Å of modeled LHVS are shown as sticks, with hydrogen bonds between the enzyme and the inhibitor shown as dashed lines. C, two-dimensional representation of the LHVS binding mode. LHVS is shown as black lines, whereas the TgCPL amino acids that surround it are shown as gray lines. Hydrogen bonds are shown as dashed lines. (C was created with ChemDraw 11.)

BO-LHVS labeling of TgCPL knock-out strains. A, activity-based protein profiling gel analysis of parental and knock-out strains with BO-LHVS. Parasites were incubated without (−) or with (+) 200 nm BO-LHVS, lysed, and analyzed by SDS-PAGE and laser-scanning fluorometry. Note the lack of BO-LHVS reactivity in the 30 kDa region of RHΔcpl and Ku80Δcpl. Asterisks indicate minor bands reactive with BO-LHVS in all strains. B, APB microscopy analysis of parental and knock-out strains with BO-LHVS. Newly invaded parasites were incubated with 200 nm BO-LHVS, fixed with paraformaldehyde, and viewed by fluorescence microscopy. Note the labeling of structures in RH parasites (arrows) and lack of reactivity in RHΔcpl parasites. The inset shows an enlargement of a representative tachyzoite.

Targeted deletion of TgCPL. A, schematic illustration of the TgCPL knock-out strategy. A knock-out construct consisting of ∼3 kb of 5′- and 3′-flanking sequence from the TgCPL gene appended to either side of a DHFR-TS-selectable marker cassette was transfected into RH and Ku80 parasites for double crossover gene replacement of TgCPL. The arrows indicate PCR primers used in B. B, agarose gel electorphoresis of PCR products derived from parental (RH and Ku80) and knock-out (RHΔcpl and Ku80Δcpl) strains by amplification with the indicated primers. C, immunoblot analysis of parental and knock-out strains probed with RαTgCPL. Note the absence of the TgCPL reactivity. Asterisks denote nonspecific bands. A parallel blot was probed with anti-actin as a loading control. D, indirect immunofluorescence assay of newly invaded intracellular tachyzoites showing MαTgCPL reactivity with RH and Ku80 (arrows) but lack of reactivity with RHΔcpl or Ku80Δcpl.

Structure of rTgCPL in complex with its propeptide. A, stereoview looking into the active site cleft with the left (L) domain on the left, the right (R) domain on the right, and the propetide on the top. The protease is colored blue, and the propeptide is colored green. N- and C-terminal residues of each polypeptide are labeled with the corresponding amino acid number. The catalytic triad (Cys³¹, His¹⁶⁷, and Asn¹⁸⁹) is colored magenta with side chains shown as sticks. The side chains of the cysteines comprising the five disulfide bonds are also shown as sticks. B and C, detail of the propeptide residues (Lys^176p–Lys^182p) that occupy the active site cleft. Propeptide amino acids in the cleft are shown as balls and sticks. The orientation matches that in A. B, surface representation of the occupied cleft. Substrate-binding subsites are labeled. The catalytic triad is colored magenta. C, the surface is removed, and TgCPL residues within 5 Å of propeptide residues 176p–182p are shown as sticks. D, stereoview of the electron density around the TgCPL active site. Electron density maps are calculated using the final refined model. Purple mesh is the σA-weighted 2F_o − F_c map contoured at 1.2 σ. The green mesh is the σA-weighted F_o − F_c difference map contoured at 3.2 σ, whereas the red mesh is the F_o − F_c difference map contoured at −3.2 σ. The positive difference density peak near the backbone of propeptide residue Leu^178p described under “Results” is shown. (Figs. 4 and 5 were created with PyMOL (51).)

BO-LHVS reacts with recombinant and native TgCPL. A, chemical structure of LHVS and BO-LHVS. Major chemical groups of LHVS from left to right are morpholinurea leucine, homophenylalanine, vinyl sulfone, phenyl. For BO-LHVS, BODIPY 493/503 is substituted for morpholinurea. B, activity-based protein profiling of rTgCPL with BO-LHVS in vitro showing dose-dependent labeling of the 30-kDa active enzyme but not labeling of the heat-inactivated (HI) enzyme. rTgCPL was incubated with BO-LHVS at the indicated ratios, resolved by 12.5% SDS-PAGE, and imaged by laser-scanning fluorometry. Molecular mass markers are indicated (kDa). C, activity-based protein profiling of native TgCPL in live cells. Tachyzoites were incubated with solvent (DMSO) or BO-LHVS and lysed. Samples were either analyzed directly (left two lanes) or immunoprecipitated (IP) with MαrTgCPL or normal mouse serum (NMS) (right two lanes) before electrophoresis and imaging as in B. The arrows denote the TgCPL bands, and an asterisk indicates the 24-kDa minor labeled product. D, competition assay with LHVS. Tachyzoites were preincubated with LHVS before exposure to 200 nm BO-LHVS and analysis as above. Asterisks denote bands that are not blocked by pretreatment with LHVS. E, subcellular distribution of active TgCPL. Extracellular or replicating intracellular tachyzoites were incubated with BO-LHVS before fixation and staining with MαrTgCPL and an Alexa 592 (red)-conjugated secondary antibody. Most of the labeled structures show dual fluorescence of the active (BO-LHVS) and total (MαrTgCPL) enzyme, with the exceptions indicated by arrows.