PDBsum entry: 3f75

Hydrolase

PDB id: 3f75

Contents

Protein chains

223 a.a. *

75 a.a. *

Ligands

EDO

Metals

_CL ×2

_BR

Waters ×167

* Residue conservation analysis

PDB id:

3f75

Name:

Hydrolase

Title:

Activated toxoplasma gondii cathepsin l (tgcpl) in complex w propeptide

Structure:

Cathepsin l protease. Chain: a. Fragment: unp residues 199-422. Synonym: toxopain-2. Engineered: yes. Cathepsin l propeptide. Chain: p. Fragment: unp residues 105-198. Synonym: toxopain-2.

Source:

Toxoplasma gondii. Organism_taxid: 383379. Strain: rh. Gene: cpl. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.99Å R-factor: 0.167 R-free: 0.206

Authors:

E.T.Larson,E.A.Merritt,Medical Structural Genomics Of Pathog Protozoa (Msgpp)

Key ref:

E.T.Larson et al. (2009). Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl. J Biol Chem, 284, 26839-26850. PubMed id: 19596863 DOI: 10.1074/jbc.M109.003780

Date:

07-Nov-08 Release date: 25-Nov-08

Protein chain

Q6DMN0  (Q6DMN0_TOXGO) -  Cathepsin L

Seq: 422 a.a.

Struc: 223 a.a.

Protein chain

Q6DMN0  (Q6DMN0_TOXGO) -  Cathepsin L

Seq: 422 a.a.

Struc: 75 a.a.

Enzyme reactions

• Enzyme class: Chains A, P: E.C.1.3.1.74 - 2-alkenal reductase.
• Reaction: n-alkanal + NAD(P)(+) = alk-2-enal + NAD(P)H

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

 Gene Ontology (GO) functional annotation 

• Biological process: proteolysis (1 term)
• Biochemical function: cysteine-type peptidase activity (2 terms)

Added reference

DOI no: 10.1074/jbc.M109.003780

J Biol Chem 284:26839-26850 (2009)

PubMed id: 19596863

Toxoplasma gondii cathepsin L is the primary target of the invasion-inhibitory compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl.

E.T.Larson, F.Parussini, M.H.Huynh, J.D.Giebel, A.M.Kelley, L.Zhang, M.Bogyo, E.A.Merritt, V.B.Carruthers.

ABSTRACT

The protozoan parasite Toxoplasma gondii relies on post-translational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 A resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.

Selected figure(s)

Figure 2.
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.

Figure 4.
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 (Cys^31, His^167, and Asn^189) 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.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 26839-26850) copyright 2009.

Figures were selected by the author.