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Abstract:

This webinar, titled "Protein Structure Meets Function," features two presentations on distinct structural biology topics. The first speaker, Nika Gibrat, discusses Napon-like Proteins (NLPs) from plant pathogens, which are pore-forming toxins targeting plant plasma membranes. Her research, supported by an Instruct Eric internship, elucidates the role of glycodil inositol phosphoril theramite (GIPC) as the NLP receptor, details the pore formation mechanism (clustering, transient pores <10kDa), and compares the activity and structural flexibility of different NLPs on monocot and dicot plant membrane models. The second speaker, Peter Pvek, explores G-quadruplex (G4) structures in DNA, their biological importance in telomeres and gene promoters, and their susceptibility to oxidative stress, specifically the formation of 8-oxoguanine (8-oxoG). He demonstrates how 8-oxoG affects G4 stability, topology, and the processivity of DNA replication enzymes, showing a location-dependent impact on enzyme bypass. Both presentations highlight the intricate relationship between molecular structure and biological function, utilizing advanced techniques like NMR, X-ray crystallography, and microfluidics.

Exploring Protein Function and DNA Structure in Structural Biology

• 00:00:05 Webinar Introduction & Instruct Eric Overview: Marit Capodnik welcomes attendees to the "Protein Structure Meets Function" webinar and introduces Instruct Eric, a pan-European network for structural biology. She outlines its services, including access to high-end technology at infrastructural centers, internships, workshops, training, and R&D funding for researchers.
• 00:02:20 Nika Gibrat Introduction: Marit introduces Nika Gibrat, a researcher from the National Institute of Chemistry, Slovenia, who studies proteins from pathogenic microorganisms that attack plant membranes. Nika recently completed an Instruct Eric-funded internship at EMBL Grenoble.
• 00:04:12 Nika's Instruct Eric Experience: Nika shares her positive experience with the Instruct Eric-funded internship at EMBL Grenoble, where she improved skills in sample preparation, crystallization, and computational methods for crystal structure solving. She highlights the value of high-throughput crystallization facilities and recommends the program.
• 00:05:23 Napon-like Proteins (NLPs) as Plant Pathogen Toxins: NLPs are a large family of microbial proteins (from bacteria, oomycetes, fungi) that act as effectors, causing significant crop losses (20-30% of world harvest). Prominent examples include pathogens causing potato famine.
• 00:06:57 GIPC as the NLP Receptor in Plant Membranes: Unlike animal cells, pore-forming toxins in plants are less understood. NLPs target glycodil inositol phosphoril theramite (GIPC) lipids, which are abundant in the outer leaflet of plant plasma membranes. GIPC structures, especially their variable sugar heads, are poorly understood.
• 00:08:19 NLP Binding and Conformational Changes: Studies on NLPPIA show it forms complexes with the terminal hexose unit of GIPC, leading to conformational changes like the widening of a binding crevice and a shift in magnesium ion position.
• 00:09:38 Long-standing Hypothesis on Plant Sensitivity: Historically, NLPs were thought to be toxic only to dicot plants due to the shorter GIPC head group, allowing closer interaction with the plasma membrane, unlike monocots with longer GIPC head groups.
• 00:10:35 Lab Results: GIPC Extraction & NLP Activity: Nika's lab has established plant plasma membrane lipid models using liposomes containing GIPCs. GIPCs are not commercially available and require time-consuming purification. They study NLP binding and pore formation using sedimentation assays, surface plasmon resonance, and tobacco leaf apoplast infiltration.
• 00:12:00 Correlation of Binding and Toxicity: Experiments with a toxic (NLPPIA) and non-toxic NLP confirm that binding to GIPC-containing membranes correlates with toxicity.
• 00:13:14 Characterizing Pore Formation: Transmission electron microscopy and high-speed atomic force microscopy reveal NLP clustering on the membrane and shallow insertion into the upper leaflet. Pores are transient, opening and closing, and appear smaller than 10 kilodaltons.
• 00:15:17 Microfluidics for Real-Time Observation: Using microfluidic systems and giant unilamellar vesicles (GUVs) with confocal microscopy, real-time events of NLP-membrane interactions were observed.
• 00:16:56 NLPPIA Pores on Both Dicot and Monocot Membranes: NLPPIA binds to and forms pores on both tobacco (dicot) and leek (monocot) GIPC-containing membranes, causing dye release and vesicle collapse, disproving the previous hypothesis about monocot insensitivity.
• 00:19:19 Fungal NLP (MP2) Activity: MP2, an NLP from the fungus Moniliophthora perniciosa (cacao pathogen), also forms pores on membranes but appears less aggressive, without causing vesicle collapse, though the end effect of pore formation is similar.
• 00:21:02 Structural Characterization of a New NLP: Despite 1700 NLP genes, only three crystal structures existed. Nika's lab structurally characterized an NLP from Phytophthora parasitica using NMR and X-ray crystallography.
• 00:22:06 NMR Reveals NLP Flexibility: NMR studies show inherent flexibility in specific loops (L2, L3, LC1) of the NLP, indicating conformational changes vital for interaction with partners.
• 00:23:46 Crystal Structure and Binding Crevice Size: The crystal structure fold is conserved, but the binding crevice of P. parasitica NLP is significantly smaller than that of NLPPIA.
• 00:24:35 Functional Assays Confirm Reduced Potency: Functional assays (tobacco infiltration, sedimentation, surface plasmon resonance) show that P. parasitica NLP is less potent and binds less stably/transiently compared to NLPPIA, correlating with its smaller binding crevice.
• 00:27:11 Loop Rigidity Impacts Function: Intramolecular interactions, amino acid composition, and molecular dynamics studies confirm that the loops (L2, L3, LC1) of P. parasitica NLP are more rigid, which likely hinders optimal GIPC binding and reduces toxicity.
• 00:28:40 Nika's Summary & Future Directions: The full mechanism of NLP-mediated pore formation is still unclear, but loops dictating binding crevice capacity are crucial. Diverse NLPs may vary in potency across plant species, requiring more testing. This study provides the first NLP solution structure, supporting predictions of flexible regions.
• 00:30:25 Q&A (Nika): Unanswered Questions & GIPC Heterogeneity: Nika explains that the structural information about the pore itself is missing, hindering full mechanistic understanding. The lack of commercially available, homogeneous GIPC samples is a major challenge for advanced structural studies like cryo-EM.
• 00:33:53 Q&A (Nika): GIPC Levels and Variability: GIPCs constitute about 40% of total lipids in plant plasma membranes, but their series and ratios vary significantly between different plant species and even cultivars, influencing NLP activity.
• 00:35:48 Q&A (Nika): Loop Conservation: Amino acid composition and length of binding loops vary between NLPs, contributing to different mechanisms; some contain more bulky amino acids, leading to increased rigidity.
• 00:37:09 Peter Pvek Introduction: Marit introduces Dr. Peter Pvek, also from the National Institute of Chemistry, who studies nucleic acid structures, particularly non-canonical DNA and RNA.
• 00:39:07 G-quadruplex (G4) Structures: Peter introduces G-quadruplexes (G4s), non-canonical DNA structures formed by planar arrangements of four guanines (G-quartets) connected by Hoogsteen base pairing, which then stack. They are polymorphic structures.
• 00:41:08 Biological Relevance of G4s: Once considered a curiosity, G4s are now known to be enriched in human genomes, particularly in telomeres (TTAGGG repeats) and gene promoters, where they can regulate gene expression.
• 00:42:51 Reactive Oxygen Species (ROS) and 8-oxoguanine (8-oxoG): ROS cause oxidative damage, with guanine being the most susceptible base to oxidation, forming 8-oxoguanine (8-oxoG). While cells have repair mechanisms, high ROS levels can lead to persistent lesions.
• 00:43:56 Impact of 8-oxoG on G4 Stability: 8-oxoG disrupts the G-quartet arrangement and thus the overall G4 structure. This disruption can be "fixed" in vitro by introducing a short oligo with a conjugated pyrene to stabilize the structure through stacking.
• 00:48:02 8-oxoG in Human Telomeric G4s: Peter shows that in human telomeric sequences, some G positions (e.g., G21) tolerate 8-oxoG without changing the G4 fold, while others (e.g., G10) lead to a completely different G4 topology, though often with reduced thermal stability.
• 00:50:11 8-oxoG in BCL2 Promoter G4s: In the BCL2 gene promoter sequence, 8-oxoG can cause a register shift in the G4 structure or act as a silent mutation if in a loop. Surprisingly, some 8-oxoG mutations can even increase thermal stability by forming base triads in capping structures.
• 00:53:19 DNA Processivity Experiments: G4 structures act as roadblocks for DNA replication enzymes. Peter describes primer extension experiments to study how a DNA polymerase bypasses G4 structures with or without 8-oxoG.
• 00:57:52 Location-Dependent Impact of 8-oxoG on Enzyme Bypass: The enzyme's ability to replicate through G4s is affected by 8-oxoG. If 8-oxoG is in a loop, it's easily bypassed. If it's in a G-quartet, its position (early vs. late in the sequence relative to replication direction) significantly impacts enzyme processivity, with later mutations causing greater destabilization and more full-length products.
• 01:02:19 Peter's Take-Home Message: Oxidative lesions significantly affect how enzymes process G4 structures, impacting biological processes like transcription and DNA binding.
• 01:02:41 Q&A (Peter): G4s in RNA: G4 structures can also be observed in RNA molecules, typically forming parallel structures. Their biological function in RNA is less understood compared to DNA, though they are found in UTRs and coding regions.
• 01:04:27 Q&A (Peter): G4 Sequence Conservation: The G4 sequences shown are mostly from human genes (telomeres, promoters), but G4-forming sequences are found across nearly all organisms, including bacteria, though observation in vivo is more challenging.