Project 12

Molecular dynamics simulations of membrane perforation machineries and the nuclear pore complex

Principle Investigator: Prof. Dr. Gerhard Hummer

Research Areas: Biophysics, Structural Biology

Summary

Complex machineries have evolved to mediate the transfer of large substrates across biological membranes, both into and out of cells and between cellular compartments. In P1, we propose to study key steps in the assembly and function of two archetypal pores in eukaryotes: (1) the ring-shaped cytolysin-like pores formed by gasdermin in inflammation, and (2) the nuclear pore complex (NPC) governing the transport between the nucleus and the cytosol. Aim 1 of this project is to resolve and characterize a key step in gasdermin-induced pore formation with the help of molecular dynamics simulations: the insertion of the beta-sheet structure into the membrane that then lines the wall of the pore. Gasdermin pores enable pro- inflammatory cytokine release and induce pyroptosis, a form of programmed cell death. Gasdermins recognize membranes in a lipid-specific manner, undergo major conformational changes upon membrane binding, assemble into large arcs and rings, and line the membrane pores with beta-barrel structures. To study gasdermin penetration into the membrane, we build on our studies of pore formation by bacterial cytolysins. To resolve the rare membrane insertion events, we will use path sampling methods. The simulations will allow us to test whether gasdermin penetration is cooperative, requires pre-ordered secondary structure, and involves specific lipids. As direct validation of key intermediates, we will work with P01 (Glaubitz) to characterize the structure of membrane-adhered and inserted peptides by solid- state nuclear magnetic resonance. Aim 2 focuses on the intracellular membrane pore that defines eukaryotes: the nuclear pore complex (NPC) governing the transport between the nucleus and the cytosol. Working closely with P15 (Beck, Lemke), we will assemble a detailed computational model of the NPC including the protein rings lining the pore, their anchors in the membrane of the nuclear envelope, the membrane itself, and the mesh of disordered FG repeats in the pore center. The NPC simulation model will allow us to test if the NPC is sensitive to mechanical perturbations of the nuclear envelope and if the patterned grafting of FG repeats contributes to NPC substrate selection and pore permeability. Our work builds on our integrative modeling studies of large membrane assemblies and of disordered proteins and protein condensates. The combination of molecular dynamics simulations with the electron tomography and fluorescence microscopy experiments of P15 promises to reveal nuclear transport at the molecular level and to give us a dynamic view of the NPC embedded in the nuclear envelope.