Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress

Alfredo J. Guerra*, Ou Zhang, Constance M.E. Bahr, My Hang Huynh, James DelProposto, William C. Brown, Zdzislaw Wawrzak, Nicole M. Koropatkin, Vern B. Carruthers

*Corresponding author for this work

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

Original languageEnglish (US)
Article numbere1007476
JournalPLoS pathogens
Volume14
Issue number12
DOIs
StatePublished - Dec 1 2018

Fingerprint

Tryptophan
Perforin
Membranes
Parasites
Clustered Regularly Interspaced Short Palindromic Repeats
Complement Membrane Attack Complex
Porins
Nucleic Acid Repetitive Sequences
Toxoplasma
Lipid Bilayers
Vacuoles
Alanine
Reproduction
Proteins
Fluorescence
Cell Membrane
Lipids
Mutation
Toxoplasma gondii perforin-like protein 1
Gene Editing

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

Cite this

Guerra, A. J., Zhang, O., Bahr, C. M. E., Huynh, M. H., DelProposto, J., Brown, W. C., ... Carruthers, V. B. (2018). Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress. PLoS pathogens, 14(12), [e1007476]. https://doi.org/10.1371/journal.ppat.1007476
Guerra, Alfredo J. ; Zhang, Ou ; Bahr, Constance M.E. ; Huynh, My Hang ; DelProposto, James ; Brown, William C. ; Wawrzak, Zdzislaw ; Koropatkin, Nicole M. ; Carruthers, Vern B. / Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress. In: PLoS pathogens. 2018 ; Vol. 14, No. 12.
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abstract = "Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.",
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Guerra, AJ, Zhang, O, Bahr, CME, Huynh, MH, DelProposto, J, Brown, WC, Wawrzak, Z, Koropatkin, NM & Carruthers, VB 2018, 'Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress', PLoS pathogens, vol. 14, no. 12, e1007476. https://doi.org/10.1371/journal.ppat.1007476

Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress. / Guerra, Alfredo J.; Zhang, Ou; Bahr, Constance M.E.; Huynh, My Hang; DelProposto, James; Brown, William C.; Wawrzak, Zdzislaw; Koropatkin, Nicole M.; Carruthers, Vern B.

In: PLoS pathogens, Vol. 14, No. 12, e1007476, 01.12.2018.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Structural basis of Toxoplasma gondii perforin-like protein 1 membrane interaction and activity during egress

AU - Guerra, Alfredo J.

AU - Zhang, Ou

AU - Bahr, Constance M.E.

AU - Huynh, My Hang

AU - DelProposto, James

AU - Brown, William C.

AU - Wawrzak, Zdzislaw

AU - Koropatkin, Nicole M.

AU - Carruthers, Vern B.

PY - 2018/12/1

Y1 - 2018/12/1

N2 - Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

AB - Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APCβ domain within the CTD reveals an unusual double-layered β-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the β-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.

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