Peptide drugs targeting class B1 GPCRs can treat multiple diseases, however there remains substantial interest in the development of orally delivered non-peptide drugs. Here we reveal unexpected overlap between signalling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist, PF 06882961, and GLP-1 that was not observed for another compound, OWL-833. Both compounds are currently in clinical trials for treatment of type 2 diabetes. High resolution cryo-EM structures reveal the binding sites for PF-06882961 and GLP-1 substantially overlap, whereas OWL-833 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not OWL-833, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of nonpeptide agonists targeting class B GPCRs.
Bacteria have mechanisms to export proteins for diverse purposes, including colonization of hosts and pathogenesis. A small number of archetypal bacterial secretion machines have been found in several groups of bacteria and mediate a fundamentally distinct secretion process. Perhaps erroneously, proteins called 'autotransporters' have long been thought to be one of these protein secretion systems. Mounting evidence suggests that autotransporters might be substrates to be secreted, not an autonomous transporter system. We have discovered a new translocation and assembly module (TAM) that promotes efficient secretion of autotransporters in proteobacteria. Functional analysis of the TAM in Citrobacter rodentium, Salmonella enterica and Escherichia coli showed that it consists of an Omp85-family protein, TamA, in the outer membrane and TamB in the inner membrane of diverse bacterial species. The discovery of the TAM provides a new target for the development of therapies to inhibit colonization by bacterial pathogens.
Highlights d Cryo-EM structure reveals how CRF1R interacts with CRF and the Gs signaling protein d Cryo-EM structure reveals interactions of Pac1nR with PACAP-38 and Gs d Evolutionary related GPCRs have greater conservation in peptide and G protein binding
Adrenomedullin (AM)
and calcitonin gene-related peptide (CGRP)
receptors are critically important for metabolism, vascular tone,
and inflammatory response. AM receptors are also required for normal
lymphatic and blood vascular development and angiogenesis. They play
a pivotal role in embryo implantation and fertility and can provide
protection against hypoxic and oxidative stress. CGRP and AM receptors
are heterodimers of the calcitonin receptor-like receptor (CLR) and
receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as
RAMP2 or RAMP3 (AM1R and AM2R, respectively).
However, the mechanistic basis for RAMP modulation of CLR phenotype
is unclear. In this study, we report the cryo-EM structure of the
AM1R in complex with AM and Gs at a global resolution of
3.0 Å, and structures of the AM2R in complex with
either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3
Å, respectively. The structures reveal distinctions in the primary
orientation of the extracellular domains (ECDs) relative to the receptor
core and distinct positioning of extracellular loop 3 (ECL3) that
are receptor-dependent. Analysis of dynamic data present in the cryo-EM
micrographs revealed additional distinctions in the extent of mobility
of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting
the TM helix and the ECD supports a role for this segment in controlling
receptor phenotype. Moreover, a subset of the motions of the ECD appeared
coordinated with motions of the G protein relative to the receptor
core, suggesting that receptor ECD dynamics could influence G protein
interactions. This work provides fundamental advances in our understanding
of GPCR function and how this can be allosterically modulated by accessory
proteins.
In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation assembly module (the TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by Quartz Crystal Microbalance with Dissipation (QCM-D) and Magnetic Contrast Neutron Reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here, we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.
Bacterial outer membrane proteins require the beta-barrel assembly machinery (BAM) for their correct folding and function. The central component of this machinery is BamA, an Omp85 protein that is essential and found in all Gram-negative bacteria. An additional feature of the BAM is the translocation and assembly module (TAM), comprised TamA (an Omp85 family protein) and TamB. We report that TamA and a closely related protein TamL are confined almost exclusively to Proteobacteria and Bacteroidetes/Chlorobi respectively, whereas TamB is widely distributed across the majority of Gram-negative bacterial lineages. A comprehensive phylogenetic and secondary structure analysis of the TamB protein family revealed that TamB was present very early in the evolution of bacteria. Several sequence characteristics were discovered to define the TamB protein family: A signal-anchor linkage to the inner membrane, beta-helical structure, conserved domain architecture and a C-terminal region that mimics outer membrane protein beta-strands. Taken together, the structural and phylogenetic analyses suggest that the TAM likely evolved from an original combination of BamA and TamB, with a later gene duplication event of BamA, giving rise to an additional Omp85 sequence that evolved to be TamA in Proteobacteria and TamL in Bacteroidetes/Chlorobi.
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