Cryo-EM structure of cell-free synthesized human histamine 2 receptor/Gs complex in nanodisc environment

Abstract Here we describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion of the receptor into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique

Structure of the HOPS tethering complex, a lysosomal membrane fusion machinery

Lysosomes are essential for cellular recycling, nutrient signaling, autophagy, and patho-genic bacteria and viruses invasion. Lysosomal fusion is fundamental to cell survival and requiresHOPS, a conserved heterohexameric tethering complex. On the membranes to be fused, HOPSbinds small membrane- associated GTPases and assembles SNAREs for fusion, but how the complexfulfills its function remained speculative. Here, we used cryo-electron microscopy to reveal the struc-ture of HOPS. Unlike previously reported, significant flexibility of HOPS is confined to its extremities,where GTPase binding occurs. The SNARE-binding module is firmly attached to the core, therefore,ideally positioned between the membranes to catalyze fusion. Our data suggest a model for howHOPS fulfills its dual functionality of tethering and fusion and indicate why it is an essential part ofthe membrane fusion machinery

Cryo-EM structure of a cell-free synthesized human histamine 2 receptor/ Gs complex in nanodisc environment

We describe the cryo-electron microscopy structure of the human histamine 2 receptor (H2R) in an active conformation with bound histamine and in complex with Gs heterotrimeric protein at an overall resolution of 3.4 Å. The complex was generated by cotranslational insertion into preformed nanodisc membranes using cell-free synthesis in E. coli lysates. It is the first structure obtained by this detergent-free strategy and the first GPCR/Gs complex structure in lipid environment. Structural comparison with the inactive conformation of H2R and the inactive and Gq-coupled active state of H1R together with structure-guided functional experiments reveal molecular insights into the specificity of ligand binding and G protein coupling for this receptor family. We demonstrate lipid-modulated folding of cell-free synthesized H2R, its agonist-dependent internalization and its interaction with endogenously synthesized H1R and H2R in HEK293 cells by applying a recently developed nanotransfer technique