Home India Study from IISC India Reveals Role of Flexibility in Pore-Forming Toxins

Study from IISC India Reveals Role of Flexibility in Pore-Forming Toxins

Study from IISC India Reveals Role of Flexibility in Pore-Forming Toxins

Researchers from the Indian Institute of Science (IISC) used all-atom molecular dynamics simulations and free energy computations to investigate the conformational changes in the pore-forming toxin Cytolysin A (ClyA) during pore formation on mammalian cell membranes.

The team found that an unfolded beta-tongue motif is an on-pathway intermediate during the protomer’s transition to the helix-turn-helix motif. They also found that point mutations in the membrane-binding motifs of the ClyA protein reduced its structural flexibility and stability, leading to a decrease in its lytic activity. The study suggests that inherent flexibility in membrane-binding proteins may play a wider role in conformational transitions and bistability in pore-forming toxins.

Summary

  • Researchers from the Indian Institute of Science (IISC) used all-atom molecular dynamics simulations and free energy computations to investigate the conformational changes in the pore-forming toxin Cytolysin A (ClyA) during pore formation on mammalian cell membranes.
  • They found that an unfolded beta-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer.
  • Point mutations in the membrane-binding motifs of the ClyA protein reduced its structural flexibility and stability, leading to a decrease in its lytic activity.
  • The study suggests that inherent flexibility in membrane-binding proteins may play a wider role in conformational transitions and bistability in pore-forming toxins.

A team of researchers from the Indian Institute of Science (IISC) used all-atom molecular dynamics simulations and free energy computations to investigate the conformational changes in the pore-forming toxin Cytolysin A (ClyA) during pore formation on mammalian cell membranes. The researchers found that an unfolded beta-tongue motif is an on-pathway intermediate during the protomer’s transition to the helix-turn-helix motif. They also discovered that point mutations in the membrane-binding motifs of the ClyA protein reduced its structural flexibility and stability, leading to a decrease in its lytic activity. The study suggests that inherent flexibility in membrane-binding proteins may play a wider role in conformational transitions and bistability in pore-forming toxins. The study connected the folding and unfolding of the protein with the pore formation activity as observed in bulk vesicle and erythrocyte lysis experiments, which is a major challenge in the field. The researchers have highlighted the importance of flexibility in the key membrane interrogating motifs of the monomer state and its correlation with the lytic activity. This research is the first to provide direct evidence for the unfolding of the tongue in the monomer as an initial step in the conformational transition and its correlation with pore formation. This could pave the way for new approaches to combat bacterial infections mediated by pore-forming toxins.

What this paper is about

  • The challenge lies in obtaining the free energy landscape associated with the conformational change for the large ClyA protein and variations induced by the point mutations and obtaining the ability to connect these changes to pore-forming activity as observed in bulk vesicle and erythrocyte lysis experiments.
  • The researcher analyzed the differential unfolding rates from the thermal unfolding simulations and observed a greater unfolding tendency in the tongue for the WT and D74 mutants compared to the Y27 and Y178 mutants that involve the main membrane-bound motifs.
  • Free energy computations of the membrane-inserted -tongue motif, combined with thermal unfolding simulations of the full monomer, provide compelling evidence to suggest that the transition to a helix-turn-helix motif of the ClyA oligomeric pore state proceeds via a key disordered -tongue intermediate that follows membrane binding.
Conformational Flexibility Is a Key Determinant for the Lytic Activity of the Pore-Forming Protein, Cytolysin by A Avijeet Kulshrestha, Satyaghosh Maurya, Twinkle Gupta, Rahul Roy, Sudeep N Punnathanam, and K. Ganapathy Ayappa

About Research Work

  • At 400 K, the -tongue RMSD is higher for the WT and D74A mutant when compared with the other mutants.
  • However, the secondary structure changes are significantly lowered in the case of the Y178F mutant indicating that the mutant shows a distinct resistance to unfolding when compared with the WT. These observations for the membrane-inserted simulations at 310 K correlate extremely well with the enhanced flexibility and increased secondary structure changes observed in the thermal unfolding simulations at 400 K for the WT compared to the Y178F mutant.
  • All-atom MD simulations and string method-based free energy computation of the transition in the membrane provide for the first time, direct evidence for the unfolding of the tongue in the monomer as an initial step in the conformational transition. This tendency for the -tongue to unfold is observed in simulations of the full monomer and the membrane-inserted -tongue motif.
  • This work assesses the initial unfolding of the monomer and its mutants to study the link between protein flexibility and the resulting unfolding and finally correlate with pore formation as observed in the lytic activity in the experiments with vesicles and erythrocytes.
  • Combined free energy and thermal unfolding simulations illustrate that flexibility in the membrane interacting motifs of the monomer state allows a loss of secondary structure to sample an on-pathway unfolded intermediate.
  • Thermal unfolding molecular dynamics simulations of monomer and its point mutants reveal a direct link between the loss of flexibility in key membrane interrogating motifs with reduced lytic ability, as revealed in both erythrocyte lysis and vesicle leakage experiments with ClyA.

Q: What did researchers from the Indian Institute of Science (IISC) investigate in their study of the pore-forming toxin Cytolysin A (ClyA)?

A: Researchers from the Indian Institute of Science (IISC) used all-atom molecular dynamics simulations and free energy computations to investigate the conformational changes in the pore-forming toxin Cytolysin A (ClyA) during pore formation on mammalian cell membranes.

Q: What did the researchers find about the beta-tongue motif in the pore-forming toxin Cytolysin A (ClyA)?

A: The researchers found that an unfolded beta-tongue motif is an on-pathway intermediate during the transition to the helix-turn-helix motif of the protomer.

Q: How did point mutations in the membrane-binding motifs of the ClyA protein affect its activity?

A: Point mutations in the membrane-binding motifs of the ClyA protein reduced its structural flexibility and stability, leading to a decrease in its lytic activity.

Q: What is this study’s significance in understanding pore-forming toxins?

A: The study suggests that inherent flexibility in membrane-binding proteins may play a wider role in conformational transitions and bistability in pore-forming toxins. The study connected the folding and unfolding of the protein with the pore formation activity as observed in bulk vesicle and erythrocyte lysis experiments, which is a major challenge in the field. The researchers have highlighted the importance of flexibility in the key membrane interrogating motifs of the monomer state and its correlation with the lytic activity. This research is the first to provide direct evidence for the unfolding of the tongue in the monomer as an initial step in the conformational transition, and it’s correlation with pore formation. This could pave the way for new approaches to combat bacterial infections mediated by pore-forming toxins.

Previous articleUnion Minister Launches Geospatial Hackathon to Promote Research, Innovation and Startups in India
Next articleARI Pune Researchers Developed New Bandage Can Rapidly Minimize Blood Loss from Wounds

LEAVE A REPLY

Please enter your comment!
Please enter your name here