It has been quite challenging to gain high resolution structural insights into an intact class B G protein-coupled receptor, despite previous solution of multiple structures for the two dominant domains, the extracellular domain (ECD) and the transmembrane helical bundle domain (TMD) of this family of receptors. Zhang et al. (1) now report a crystal structure of full length glucagon receptor (GCGR) in an inactive conformation stabilized by the non-peptidyl antagonist, NNC0640, and mAb1, bound to the ECD. In this new structure, the ECD is elongated above the TMD, with mAb1 resting on extracellular loop 1 (ECL1), and with the stalk region that links the two dominant receptor domains present in a β-strand conformation lying across the helical bundle between ECL1 and ECL2/ECL3. Of particular interest, hydrogen bonds are formed between the stalk and ECL1 to establish a compact β-sheet. The conformation of the stalk in this structure is different from the α-helical extension of TM1 present in the previous solved structure of the isolated ECD of this receptor (2), with the orientation of the ECD in the new structure quite different from that previously predicted. The authors used data from hydrogen-deuterium exchange, disulfide crosslinking, and molecular dynamics to suggest that the relatively stable β-sheet formed by the stalk and ECL1 plays an important role in controlling accessibility of the orthosteric peptide ligand to its site of docking and in the transition of inactive to active receptor states. A hypothetical model is proposed whereby the C-terminus of glucagon gains access to the peptide-binding groove within the ECD, a step that requires ECD separation from the stalk/ECL1 complex, with this initial ligand-binding event leading to a conformational change in the receptor that is not yet understood, allowing the N-terminus of glucagon to dock within the TMD to activate the receptor. A recent report of the use of cryo-EM to determine the structure of another member of the class B family, the calcitonin receptor, in active conformation in complex with salmon calcitonin and its heterotrimeric G protein (3), also emphasizes the relative mobility of ECD and TMD, and the importance of dynamic changes in orientation of these domains. It will be important to gain more insights into the structure and conformational flexibility of apo-receptors in this family to better understand how the natural peptide ligands gain access to the ECD, and to learn more about other possible sites of contact between ECD and TMD that could contribute to conformational changes in the TMD. These reports emphasize the functional importance and likely variations that will exist in the relative orientations of these key structural domains for this class of GPCRs.
 Zhang et al. (2017). Structure of the full-length glucagon class B G-protein-coupled receptor. Nature, doi:10.1038/nature222363. [PMID: 28514451]
 Siu et al. (2013). Structure of the human glucagon class B G-protein-coupled receptor. Nature, 488:444-449. [PMID: 23863937]
 Liang et al. (2017). Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature,doi: 10.1038/nature22327. [Epub ahead of print] [PMID: 28437792]
Comments by Laurence J. Miller (Mayo Clinic, Scottsdale, AZ, USA)
Tagged with: GPCRs
Posted in Hot Topics
The glucagon-like peptide-1 receptor (GLP-1R) is a major target for treatment of Type 2 diabetes but has been refractory to the development of small molecule compounds as potential therapeutics. Song et al., (1) report the first crystal structures of the GLP-1R transmembrane domain in complex with 2 distinct negative allosteric modulators (NAMs) (PF-06372222 and NNC0640). The work provides insight into inactive state structure for the GLP-1R and key interactions that drive inhibitor potency. Moreover, the work allows modelling of an allosteric agonist (and positive allosteric modulator, PAM), Novo Nordisk compound 2, to reveal a potential mechanism for allosteric receptor activation that is supported by mutagenesis and molecular dynamics simulations. The proposed mechanism would lead to a decreased energy barrier for receptor activation through reorganisation of hydrogen bond networks at the base of the receptor that are important for receptor quiescence, and is consistent with the known pharmacology of the PAM in modulating orthosteric peptide activation of the receptor. This work, combined with novel structures of active class B receptors, opens up new possibilities for design of small molecule compounds to manipulate receptor pharmacology and as potential therapeutic drug leads.
 Song et al. (2017) Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators. Nature, doi:10.1038/nature22378. [PMID 28514449]
Comments by Patrick Sexton (Monash Univeristy, Melbourne)
We are pleased to announce the first, public, beta-release of the Guide to IMMUNOPHARMACOLOGY (GtoImmuPdb). The GtoImmuPdb is a Wellcome Trust-funded extension to the existing Guide to PHARMACOLOGY (GtoPdb) and the beta-release (v1.0) marks an important milestone in its production and development. GtoImmuPdb aims to provide improved data exchange between immunology and pharmacology expert communities, so to better support research and development of drugs targeted at modulating immune, inflammatory or infectious components of disease. The underlying GtoPdb schema has been extended to incorporate new immune system specific data types (such as processes, cell types and disease) and the GtoPdb website has been developed to surface this new data and incorporate it into the existing search and browse mechanisms. A new Guide to IMMUNOPHARMACOLOGY portal (Figure 1)(www.guidetoimmunopharmacology.org) has been developed, which serves as a unique immunological access-point to the Guide to PHARMACOLOGY.
The GtoImmuPdb enriches the existing GtoPdb by flagging targets and ligands of immunological relevance and linking these targets to immunological process, cell types and relevant diseases. In terms of processes and cell types, GtoImmuPdb has developed top-level categories (Figure 2), that aim to be meaningful and intuitive to immunologists, against which targets and ligands in the database can be annotated. These categories are underpinned by the use of both the Gene Ontology and the Cell Ontology. Using recognised ontologies provides a controlled vocabulary for higher resolution annotation (Figure 3). It also facilitates interoperability between new data types in GtoImmuPdb and external resources that also use these ontologies.
Data linking targets and ligands to disease is also incorporated into GtoImmuPdb, with the curation of disease associations using resources such as OrphaNet, Disease Ontology and OMIM (Figure 4).
As well as the development of the GtoImmuPdb Portal, the web-interface has been further developed with immunological data and users in mind. It has been designed to provide a unique ‘GtoImmuPdb view’ of the data, highlighting content of immunological relevance and prioritising immunological data in search results and display. It includes features that highlight targets, target families and ligands of immunological relevance (Figure 5); toggle buttons to enable the GtoImmuPdb view to be switched on and off (Figure 6); and new pages and sections to display immunological data (Figure 7).
Development of the beta-release is ongoing with regular updates planned over the next few months as the quantity of data captured increases and improvements in the site layout and function are made. One of our priorities over the next 6 months is to undertake rigorous site testing with interested user groups to capture more insight and feedback. We welcome those interested and potential future users to get in touch with us.
This project is supported by a 3-year grant awarded to Professor Jamie Davies at the University of Edinburgh by the Wellcome Trust (WT).
Our 4th database release of 2017 was published on 23rd May 2017. It now includes 15133 interactions between 2813 targets and 8900 ligands. For full release statistics see the Guide to PHARMACOLOGY About page.
Updates have been made to the following target families:
The Guide to IMMUNOPHARMACOLOGY
The most major update in this release is the inclusion of the first beta-release of the Guide to IMMUNOPHARMACOLOGY (GtoImmuPdb).
GtoImmuPdb is a Wellcome Trust-funded extension to the existing Guide to PHARMACOLOGY (GtoPdb) and this release marks an important milestone in its production and development. GtoImmuPdb aims to provide improved data exchange between immunology and pharmacology expert communities, so to better support research and development of drugs targeted at modulating immune, inflammatory or infectious components of disease. The underlying GtoPdb schema has been extended to incorporate new immune system specific data types (such as processes, cell types and disease) and the GtoPdb website has been developed to surface this new data and incorporate it into the existing search and browse mechanisms. A new Guide to IMMUNOPHARMACOLOGY portal (www.guidetoimmunopharmacology.org) has been developed, which serves as a unique immunological access-point to the Guide to PHARMACOLOGY.
For more information, please see our separate blog post on the Guide to IMMUNOPHARMACOLOGY beta-release.
Ligand activity graphs
In our 2017.2 release we announced the development of ligand activity graphs which can be used to compare activity ranges across species using data extracted from GtoPdb and ChEMBL.
These have recently been updated to use the latest ChEMBL release (ChEMBL_23), which was made available on 19th May 2017. We have also applied a few minor bug fixes to the graphs, most notably with the box plot display. We are very interested in getting feedback on this new feature and would encourage anyone who has used it or looked at it to get in touch with comments.
The latest GtoPdb release has also incorporated changes to our news and hot topics pages. The revised news page has been designed to give greater prominence to our blog feed, which is now our main source of news.
Our hot topics page has also been modified to better capture and present interesting topics in pharmacology. It now presents a more refined list of the latest hot topics, with link from selected ones to our blog, where contributors have provided more detailed and enriched comments.
Molecular details of receptor activation remain scarce when it comes to Class Frizzled receptors and even less is known about the dynamics between the N-terminal cysteine rich domain (CRD) and the transmembrane domain (TMD) in the absence and presence of ligand. The Class Frizzled comprises 10 isoforms of FZDs (FZD1-10) and Smoothened (SMO), which mediate WNT and Hedgehog signalling respectively. The recently published structure of a full-length SMO bound to the stabilizing compound TC114 builds on emerging concepts from earlier crystal structures of SMO and provides novel insight into how structural rearrangements of the CRD relative to the receptor core coordinate receptor activation while relating this to WNT receptors (1). The ligand-dependent communication between the CRD and the TMD is of special interest because of the known requirement of Class Frizzled receptors to bind endogenous agonists with their CRD. The observed movement of the extracellular extension of SMO-TM6 and extracellular loop 3 suggests a bimodal binding of the agonist to the CRD and the TMD as has been seen for Class B receptors. While this study focuses on the ligand-dependent structural rearrangements on the extracellular part of SMO, it remains unresolved how the conformational changes on the extracellular side of Class Frizzled receptors relate to activating movements of the transmembrane helices on the intracellular side – leaving ample room for future discoveries. The present study opens a new chapter in drug discovery through the use of structure- and mechanism-based drug design to fuel ideas and hopes of successfully targeting FZDs by small molecule drugs. On a more general scale, this work published in Nature Communications by the groups of Fei Xu, Wenfu Tan, Houchao Tao and Raymond Stevens contributes to a better understanding of the role of large extracellular domains in GPCRs with regard to ligand recognition and the engagement of the transmembrane core of the receptor for signal initiation.
 Zhang X et al. (2017). Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand. Nat Commun., 8:15383. [PMID:28513578].
Comments by Shane C. Wright & Gunnar Schulte (Karolinska Institutet)
Ca2+-ATPases are members of the P-type transporters and are usually regarded as post-stimulus recovery mechanisms, allowing calcium ions to be removed from the cytosol either via re-accumulation into sarcoplasmic/endoplasmic reticulum (the SERCA pumps) or extrusion outside the cell (the PMCA pumps). Here, Norimatsu and colleagues (1) have used X-ray solvent contrast modulation to assess density maps of four activation states of the SERCA1, focussing on the interaction between the ten transmembrane helices and their surrounding phospholipid environment. Their observations suggest an unexpected movement of the transporter core, which allows an exaggerated ‘waving’ of the cytoplasmic-extended calcium-binding domain during the cycle of calcium transport.
 Norimatsu et al. (2017). Protein-phospholipid interplay revealed with crystals of a calcium pump. Nature 545(7653):193-198. [PMID:28467821]
Comments by Steve Alexander (@mqzspa)
The last 10 years has seen a huge increase in the numbers of X-ray structures solved for GPCRs. However solving fully active structures in complex with G proteins remains challenging. Recent advances in cryo-electron microscopy (cryo-EM), including improved direct electron detectors and image processing methods, mean that near atomic resolution is becoming possible for protein complexes of smaller molecular size (sub 100kDa). Cryo-EM has the advantage over X-ray crystallography in that structures are determined from single molecules, avoiding the need to grow crystals (although sample preparation is still not trivial). This is particularly useful for large macromolecular complexes or proteins with labile domains which are difficult to crystallize. A dramatic step up in quality of data collected has been achieved using the volta phase plate which greatly increases the contrast, particularly at the lower end of the molecular size range, meaning that structures of GPCR complexes are now within reach. The first example of this has been published in Nature by the groups of Patrick Sexton at Monash and Wolfgang Baumeister at the Max Planck in Martinsried. Using the volta phase plate the cryo-EM structure of the full length calcitonin receptor bound to a peptide agonist in complex with the heterotrimeric G protein was solved to 3.8 Ä. The structure reveals the conformational changes which occur upon activation of a Class B GPCR. Cryo-EM is set to revolutionise structural biology and with the rapid progress is now encompassing GPCR complexes. We expect others to follow in the near future.
 Liang et al. (2017). Phase-plate cryo-EM structure of a class B GPCR–G-protein complex. Nature, doi: 10.1038/nature22327. [PMID: 28437792]
Comments by Dr. Fiona H. Marshall (Director & CSO, Heptares Therapeutics) (@aston_fm)