Activation and Inactivation Mechanisms of Membrane-bound Guanylyl Cyclases

• Applicant: Professor Dr. Elmar Behrmann
• Subject Area: Structural Biology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 405360871

How do life forms sense their environment? On the cellular level, both G-protein coupled receptors (GPCRs) and membrane-bound guanylyl cyclases (mGCs) play key roles in detecting extracellular signals, and relaying them to the inside of the cell. Different from GPCRs, where multiple proteins are involved in signal transduction, -modulation and -amplification, mGCs have receptor, modulator and effector domains combined into a single protein. Different from GPCRs, no high-resolution structural data is available for full-length mGCs. Consequently, we still do not know the molecular mechanisms that allow mGCs to carry out all three tasks. Core subject of this project proposal is thus determining the 3D structure of a full-length mGC using single-particle cryo-electron microscopy (cryo-EM), and gaining understanding of the molecular mechanisms of its activation and inactivation. Distinguishing feature of the project is that I will make full use of sea urchin sperm as a model system: not only do we have good biochemical understanding of signal transduction by mGC in this system. The fact that sea urchin sperm must be able to trace individual molecules of chemo-attractants to find an egg has resulted in the evolution of flagella that are densely packed with the receptor mGC. My plan is to use sperm of the sea urchin Arbacia punctulata to isolate enough material for structure determination by cryo-EM and single particle reconstruction. I will also induce defined functional states of the A. punctulata mGC and determine their structures to uncover the molecular mechanism of its activation and inactivation. Specifically, I plan to uncover how and in which stoichiometry the chemo-attractant resact is bound by the mGC complex; investigate which structural rearrangements are induced by ligand-binding in the extracellular domain; understand how extracellular and intracellular domains are structurally and functionally coupled; and unveil the structural and functional aspects of mGC modulation by phosphorylation and dephosphorylation. Phylogenetic comparison as well as conserved phosphorylation sites indicate that A. punctulata mGC is an ortholog of mammalian peptide-binding GC-A and GC-B. Therefore, these data will not only give us fundamental insights into the molecular mechanisms of chemo-attractant sensing in A. punctulata sperm cells, but also shed light on the molecular mechanism of mGC-based hormone signal transduction in eukaryotes.

• DFG Programme: Research Grants