ABSTRACT
Toxoplasmosis, caused by Toxoplasma gondii, poses risks to vulnerable populations. TgPDCD5, a secreted protein of T. gondii, induces apoptosis through heparan sulfate-mediated endocytosis. The entry mechanism of TgPDCD5 has remained elusive. Here, we present the solution structure of TgPDCD5 as a helical bundle with an extended N-terminal helix, exhibiting molten globule characteristics. NMR perturbation studies reveal heparin/heparan sulfate binding involving the heparan sulfate/heparin proteoglycans-binding motif and the core region, influenced by proline isomerization of P107 residue. The heterogeneous proline recruits a cyclophilin TgCyp18, accelerating interconversion between conformers and regulating heparan/heparin binding. These atomic-level insights elucidate the binary switch's functionality, expose novel heparan sulfate-binding surfaces, and illuminate the unconventional cellular entry of pathogenic TgPDCD5.
ABSTRACT
Arsenite-induced stress granule (SG) formation can be cleared by the ubiquitin-proteasome system aided by the ATP-dependent unfoldase p97. ZFAND1 participates in this pathway by recruiting p97 to trigger SG clearance. ZFAND1 contains two An1-type zinc finger domains (ZF1 and ZF2), followed by a ubiquitin-like domain (UBL); but their structures are not experimentally determined. To shed light on the structural basis of the ZFAND1-p97 interaction, we determined the atomic structures of the individual domains of ZFAND1 by solution-state NMR spectroscopy and X-ray crystallography. We further characterized the interaction between ZFAND1 and p97 by methyl NMR spectroscopy and cryo-EM. 15N spin relaxation dynamics analysis indicated independent domain motions for ZF1, ZF2, and UBL. The crystal structure and NMR structure of UBL showed a conserved ß-grasp fold homologous to ubiquitin and other UBLs. Nevertheless, the UBL of ZFAND1 contains an additional N-terminal helix that adopts different conformations in the crystalline and solution states. ZFAND1 uses the C-terminal UBL to bind to p97, evidenced by the pronounced line-broadening of the UBL domain during the p97 titration monitored by methyl NMR spectroscopy. ZFAND1 binding induces pronounced conformational heterogeneity in the N-terminal domain of p97, leading to a partial loss of the cryo-EM density of the N-terminal domain of p97. In conclusion, this work paved the way for a better understanding of the interplay between p97 and ZFAND1 in the context of SG clearance.
Subject(s)
Intracellular Signaling Peptides and Proteins , Models, Molecular , Stress Granules , Valosin Containing Protein , Humans , Arsenites/metabolism , Arsenites/chemistry , Crystallography, X-Ray , Protein Binding , Protein Domains , Stress Granules/metabolism , Ubiquitin/metabolism , Valosin Containing Protein/metabolism , Valosin Containing Protein/chemistry , Valosin Containing Protein/genetics , Zinc Fingers , Protein Folding , Magnetic Resonance Imaging , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolismABSTRACT
Toxoplasmosis is a systematic protozoan disease caused by a tiny parasite Toxoplasma gondii. The infection can be dangerous for pregnant woman and people with weak immune systems. The secreted protein named TgPDCD5 (Programmed cell death protein 5 from Toxoplasma gondii) plays an important role in apoptosis-inducing effect on host cells. Here, we report the 1H, 13C, and 15N resonance assignments of TgPDCD5. This work provides the ground for further structural elucidate and biophysical investigation about protein function.
Subject(s)
Apoptosis Regulatory Proteins/analysis , Nuclear Magnetic Resonance, Biomolecular , Protozoan Proteins/analysis , Toxoplasma/metabolism , Nitrogen Isotopes , Protein Structure, Secondary , Proton Magnetic Resonance SpectroscopyABSTRACT
A sperm motility inhibitor isolated from porcine seminal plasma is identical to porcine beta-microseminoprotein (MSP). Circular dichroism (CD) and nuclear magnetic resonance (NMR) data showed that the native and recombinant porcine MSPs exhibit very similar structure. The five disulfide pairings on porcine MSP were unambiguously assigned based on NMR data and further confirmed using structural calculations. Surprisingly, our derived pairings differ from those recently reported for ostrich MSP based on matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis. Furthermore, the secondary structure was determined to comprise one four-stranded and two double-stranded antiparallel beta-sheets. As we know, this is the first detailed secondary structure reported among several types of MSPs.
