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One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway., eLife, 8, ISSN:2050-084X

One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the G beta subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of G beta as scaffold for recruiting G alpha subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

Vertebrate cyclic nucleotide-gated (CNG) channels are non-selective cation channels. They play an essential role in the signal transduction of visual and olfactory sensory systems. There, CNG channels close in response to the decrease of cyclic nucleotide concentration, inducing the hyperpolarisation of the plasma membrane. In rod and cone photoreceptors, their role is crucial for the control of intracellular calcium concentration and the capability of the photoreceptors to respond to light stimuli. CNG channels mutations have been identified in patients affected by retinitis pigmentosa and achromatopsia, two common disabling vision diseases for which no cure is currently available. Discovered in the mid-80s, CNG channels have since been extensively studied. However, CNG channels have been notoriously dicult targets for structural biology, in part due to their particular subunit composition and stoichiometry. My PhD project focused on producing the first structure of a heteromeric CNG channel. In the first part of my work, I solved the structure in the closed (apo) state of the rod CNG channel (CNGA1/B1) purified from bovine retinas to 3.5 Å resolution. This allowed identifying new structural features on the regulatory CNGB1 subunit not present in homomeric CNGA channels. These include a new gate within the ion conduction pathway, that we called “CNGB gate”. This gate is composed of a single residue (R994) on the CNGB1 subunit that reaches into the ionic pathway. While this residue is conserved among all CNGB1 subunits, it is replaced by glutamine in the CNGB3 subunit (present in the cones CNG channels) in some species. I have identified one helix in the CNGB1 subunit that was not previously described. We called this helix the “D-helix”. In the second part of my work, I present how this new structural feature is involved in the CNG channel/calmodulin interaction. During the four years of my thesis, other research groups have produced structures of the rod and cone CNG channels – the human rod CNG channel (CNGA1/B1) and the human cone CNG channel (CNGA3/B3). I have analysed the similarities and differences in these structures in a review. Altogether, their publications complement and support our structural discoveries. In addition, I identified an uncharacterised density in the cryo-EM maps of the human CNGA1/B1 channel, that I attributed to calmodulin (a known regulator of rod CNG channel). Furthermore, I have obtained a density map of the bovine rod CNG channel bound to calmodulin. These results highlight the binding of the calmodulin to the D-helix of the CNGB1 subunit. Altogether, my work provides a new and significant understanding of the structural basis of the gating mechanism, ion permeation, and modulation of CNG channels. These results will ultimately inform new strategies to address questions regarding the role and implication of CNG channels in signal transduction within physiological and pathological contexts.

Interactions between emerging nascent polypeptide chains and the ribosome can modulate cotranslational protein folding. However, it has remained unclear how such interactions can affect the binding of nascent chains to their cellular targets. We thus investigated on the ribosome the interaction between two intrinsically disordered proteins of opposite charge, ACTR and NCBD, which form a high-affinity complex in a coupled folding-and-binding reaction. Using fluorescence correlation spectroscopy and arrest-peptide-mediated force measurements in vitro and in vivo, we find that the ACTR-NCBD complex can form cotranslationally but only with ACTR as the nascent chain and NCBD free in solution, not vice versa. We show that this surprising asymmetry in behavior is caused by pronounced charge interactions: attraction of the positively charged nascent chain of NCBD to the negatively charged ribosomal surface competes with complex formation and prevents ACTR binding. In contrast, the negatively charged nascent ACTR is repelled by the ribosomal surface and thus remains available for productively binding its partner. Electrostatic interactions may thus be more important for cotranslational folding and binding than previously thought.