Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

SummaryLigand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists.

Copper diacetyl-bis(N4-methylthiosemicarbazone), Cu(II)ATSM, is a promising agent for imaging hypoxic tissue. Here we present results that provide insight into the chemical and electronic properties underlying previously observed structure-activity relationships. Density functional theory (DFT) calculations on the electronic structures and molecular orbitals of a series of 13 Cu(II)bis(thiosemicarbazone) analogues with different alkylation patterns and with fixed geometries based on the known structure of Cu(II)PTSM showed that the LUMO and the next lowest orbital were very close in energy, and their energy order was strikingly dependent on the ligand alkylation pattern in a way that correlated with hypoxia-selectivity and redox potentials. The LUMOs of Cu(II)ATSM and other hypoxia-selective analogues were predominantly metal-based (leading to a singlet reduced species) while the LUMOs of Cu(II)PTSM and other nonselective analogues were predominantly ligand-based (leading to a triplet reduced species). Upon relaxation of the geometric constraint and full optimization in both Cu(II)ATSM and Cu(II)GTS, the metal-based orbital became the LUMO, and the singlet was the thermodynamically preferred form of the reduced species. Chemical and electrochemical investigation showed that all Cu(II) complexes were reducible, but Cu(I)PTSM and other nonselective analogues dissociated immediately upon reduction with release of ligand (detected by UV-vis) while Cu(I)ATSM and other hypoxia-selective analogues did not. Instead they were rapidly re-oxidized to the Cu(II) complex by molecular oxygen. The reversible electrochemical reduction of nonselective complexes Cu(II)PTSM and Cu(II)GTS became irreversible in the presence of weak acid, whereas that of Cu(II)ATSM was unaffected. In light of these results we present a model to explain the structure-activity relationships on the basis of electronic structure and molecular vibrations.