This webinar, hosted by Instruct-ERIC, features two presentations on protein-membrane interactions and DNA structural dynamics.
The first presentation, by Nika Gibrat, explores Neprilysin-like proteins (NLPs), a family of pore-forming toxins from plant pathogens. Gibrat discusses how NLPs bind to specific plant membrane lipids called GIPCs, leading to pore formation and cell damage. She presents experimental evidence showing that NLPs can form pores in both dicot and monocot model membranes, challenging previous hypotheses. Structural studies (NMR and crystallography) are used to investigate how the flexibility of the protein's binding crevice influences its toxic potency.
The second presentation, by Dr. Peter Pvek, focuses on the structural and functional consequences of oxidative damage to non-canonical DNA structures known as G-quadruplexes (G4s). Dr. Pvek explains how the common oxidative lesion, 8-oxoguanine, affects the stability and folding of G4s. His research demonstrates that the impact of this damage is highly context-dependent, sometimes destabilizing the structure and other times leading to structural rearrangements. Primer extension assays reveal how these G4 structures and their oxidized forms act as roadblocks for DNA processing enzymes, with the position of the lesion significantly influencing the enzyme's ability to bypass the structure.
Investigating Plant Toxins and Damaged DNA: Highlights from the Instruct-ERIC Webinar
00:00:05 Introduction to Instruct-ERIC: The webinar begins with an overview of Instruct-ERIC, a European research infrastructure for structural biology that provides access to high-end technology, expertise, internships, and R&D funding for researchers.
00:04:12 Nika Gibrat's Research on Plant Toxins: Nika Gibrat introduces her work on Neprilysin-like proteins (NLPs), a large family of toxins produced by plant pathogens. She also highlights her positive experience with an Instruct-ERIC funded internship at EMBL Grenoble, which advanced her skills in protein crystallization.
00:06:57 Mechanism of Action: NLPs Target GIPC Lipids: NLPs function as pore-forming toxins that target plant membranes. Their specific receptor is a class of lipids called glycosylinositol phosphorylceramides (GIPCs), which are abundant in the outer leaflet of the plant plasma membrane.
00:09:38 Challenging the Dicot-Only Toxicity Hypothesis: Gibrat explains the historical hypothesis that NLPs were only toxic to dicot plants, as the longer sugar heads on monocot GIPCs were thought to sterically hinder the toxin from reaching the membrane.
00:15:52 Experimental Proof of Monocot Activity: Using microfluidics with fluorescently labeled proteins and GUVs (Giant Unilamellar Vesicles), the research demonstrates that NLPs can successfully bind to and form pores in membranes containing GIPCs from both dicots (tobacco) and monocots (leek), overturning the previous belief.
00:26:45 Structural Basis for Toxicity: Structural analysis via NMR and X-ray crystallography reveals that the potency of an NLP is linked to the flexibility and size of its GIPC-binding crevice. A more rigid and smaller crevice, caused by intramolecular interactions in the surrounding loops, results in weaker binding and lower toxicity.
00:39:35 Dr. Peter Pvek on G-Quadruplexes (G4s): Dr. Peter Pvek introduces his research on G-quadruplexes (G4s), non-canonical four-stranded DNA structures formed from guanine-rich sequences. These structures are found in key genomic regions like telomeres and gene promoters.
00:43:00 The Impact of Oxidative Damage on G4s: Pvek explains that guanine is the most easily oxidized DNA base, leading to the formation of 8-oxoguanine (oxyG). This lesion alters the hydrogen bonding pattern and can disrupt or destabilize G4 structures.
00:48:25 Context-Dependent Structural Effects: Research shows the effect of an oxyG lesion is highly dependent on its location. Depending on the sequence and position, the lesion can either completely disrupt the G4, be tolerated with minimal change, or cause a significant structural rearrangement (e.g., a "register shift") where the DNA strand slides to exclude the damaged base from the core.
00:53:34 G4s as Roadblocks for DNA Enzymes: Using primer extension assays, Pvek demonstrates that G4 structures act as roadblocks for DNA polymerases. The presence and position of an oxyG lesion modulates this effect; a highly destabilizing lesion can make the G4 easier for the enzyme to bypass, suggesting that oxidative damage can alter the regulatory function of these structures.