Siv A. Hjorth

GPCR-Mediated Signaling of Metabolites

In addition to their bioenergetic intracellular function, several classical metabolites act as extracellular signaling molecules activating cell-surface G-protein-coupled receptors (GPCRs), similar to hormones and neurotransmitters. Signaling metabolites generated from nutrients or by gut microbiota target primarily enteroendocrine, neuronal, and immune cells in the lamina propria of the gut mucosa and the liver and, through these tissues, the rest of the body. In contrast, metabolites from the intermediary metabolism act mainly as metabolic stress-induced autocrine and paracrine signals in adipose tissue, the liver, and the endocrine pancreas. Importantly, distinct metabolite GPCRs act as efficient pro- and anti-inflammatory regulators of key immune cells, and signaling metabolites may thus function as important drivers of the low-grade inflammation associated with insulin resistance and obesity. The concept of key metabolites as ligands for specific GPCRs has broadened our understanding of metabolic signaling significantly and provides a number of novel potential drug targets.

The existence of multiple conformers of interleukin-21 directs engineering of a superpotent analogue.

The high resolution three-dimensional structure of human interleukin (hIL)-21 has been resolved by heteronuclear NMR spectroscopy. Overall, the hIL-21 structure is dominated by a well defined central four-helical bundle, arranged in an up-up-down-down topology, as observed for other cytokines. A segment of the hIL-21 molecule that includes the third helical segment, helix C, is observed to exist in two distinct and interchangeable states. In one conformer, the helix C segment is presented in a regular, α-helical conformation, whereas in the other conformer, this segment is largely disordered. A structure-based sequence alignment of hIL-21 with receptor complexes of the related cytokines, interleukin-2 and -4, implied that this particular segment is involved in receptor binding. An hIL-21 analog was designed to stabilize the region around helix C through the introduction of a segment grafted from hIL-4. This novel hIL-21 analog was demonstrated to exhibit a 10-fold increase in potency in a cellular assay.

Crystal Structure of Interleukin-21 Receptor (IL-21R) Bound to IL-21 Reveals That Sugar Chain Interacting with WSXWS Motif Is Integral Part of IL-21R

Background: The class I cytokine IL-21 exerts pleiotropic effects on innate and adaptive immunity. Results: We obtained the crystal structure of the partially glycosylated IL-21 receptor (IL-21R) bound to IL-21. Conclusion: A sugar chain is an integral part of IL-21R. Significance: This structure offers an insight into the putative role of the class I cytokine receptor signature motif. IL-21 is a class I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. It signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common γ-chain. A hallmark of the class I cytokine receptors is the class I cytokine receptor signature motif (WSXWS). The exact role of this motif has not been determined yet; however, it has been implicated in diverse functions, including ligand binding, receptor internalization, proper folding, and export, as well as signal transduction. Furthermore, the WXXW motif is known to be a consensus sequence for C-mannosylation. Here, we present the crystal structure of IL-21 bound to IL-21R and reveal that the WSXWS motif of IL-21R is C-mannosylated at the first tryptophan. We furthermore demonstrate that a sugar chain bridges the two fibronectin domains that constitute the extracellular domain of IL-21R and anchors at the WSXWS motif through an extensive hydrogen bonding network, including mannosylation. The glycan thus transforms the V-shaped receptor into an A-frame. This finding offers a novel structural explanation of the role of the class I cytokine signature motif.

Mutational analysis of the interaction of the N‐ and C‐terminal ends of angiotensin II with the rat AT1A receptor

The role of different residues of the rat AT1A receptor in the interaction with the N‐ and C‐terminal ends of angiotensin II (AngII) was studied by determining ligand binding and production of inositol phosphates (IP) in COS‐7 cells transiently expressing the following AT1A mutants: T88H, Y92H, G196I, G196W and D278E. G196W and G196I retained significant binding and IP‐production properties, indicating that bulky substituents in position 196 did not affect the interaction of AngIIs C‐terminal carboxyl with Lys199 located three residues below. Although the T88A mutation did not affect binding, the T88H mutant had greatly decreased affinity for AngII, suggesting that substitution of Thr88 by His might hinder binding through an indirect effect. The Y92H mutation caused loss of affinity for AngII that was much less pronounced than that reported for Y92A, indicating that His in that position can fulfil part of the requirements for binding. Replacing Asp278 by Glu caused a much smaller reduction in affinity than replacing it by Ala, indicating the importance of Asps β‐carboxyl group for AngII binding. Mutations in residues Thr88, Tyr92 and Asp278 greatly reduced affinity for AngII but not for Sar1 Leu8‐AngII, suggesting unfavourable interactions between these residues and AngIIs aspartic acid side‐chain or N‐terminal amino group, which might account for the proposed role of the N‐terminal amino group of AngII in the agonist‐induced desensitization (tachyphylaxis) of smooth muscles.

Mutations in transmembrane segment VIJ of the AT1 receptor differentiate between closely related insurmountable and competitive angiotensin antagonists

Chimeric constructs between the human and the Xenopus laevis AT1 receptor have demonstrated, that the binding of non‐peptide angiotensin antagonists is dependent on non‐conserved residues located deep in transmembrane segment VII of the AT1 receptor. Here we have studied four pairs of closely related antagonists each consisting of a competitive and an insurmountable compound differentiated by one out of three different types of minor chemical modifications. None of the antagonists bound to the Xenopus receptor and the binding of all of the compounds to the human receptor was severely impaired by the introduction of non‐conserved residues from transmembrane segment VII of the Xenopus receptor. In all four pairs of antagonists the competitive compound was affected more by these substitutions than the corresponding insurmountable one (209 vs. 22, 281 vs. 29, 290 vs. 29 and 992 vs. 325‐fold increase in K i values). A similar pattern was observed in response to substitution of a single non‐conserved residue in transmembrane segment VII, Asn295 to Ser. These results indicate that a common molecular mechanism distinguishes the interaction of insurmountable and competitive antagonists with the AT1 receptor.

Metal-Ion Sites as Structural and Functional Probes of Helix–Helix Interactions in 7TM Receptors

An important group of integral membrane proteins is the G-protein-coupled receptor superfamily, which today constitutes more than 100 cloned receptors. They signal information across the membrane for a diverse collection of ligand molecules ranging from, for example, metal-ions, classical transmitters, and neuropeptides to larger glycoprotein hormones.,2 Despite the large variety of ligands binding to these receptors, the seven transmembrane segments of G-proteincoupled receptors (7TM) are believed to define a common fold of presumably seven a-helices traversing the membrane in an anti-parallel way. Even though the number of solved structures of soluble proteins has been steadily increasing, there has been no similar success in the solution of membrane protein structures. As a consequence, structural information is currently pursued mainly by noncrystallographic methods. Hence, for the 7TM receptor superfamily, detailed information on the receptor structure is scarce, and the only real structural information available is the electron projection density maps of bovine and frog r h ~ d o p s i n ~ . ~ and the iow-resolution three-dimensional data for rhodop~ in .~ With the limited resolution of these structures, the assignment of individual helices to the electron density remains and the detailed understanding of the intramolecular signal transduction mechanism still remains obscure. Important progress does, however, appear to have been made in recent years in respect of some general overall molecular

Engineering of metal-ion sites as distance constraints in structural and functional analysis of 7TM receptors

G-protein-coupled receptors with their seven transmembrane (7TM) segments constitute the largest superfamily of proteins known. Unfortunately, still only relatively low resolution structures derived from electron cryo-microscopy analysis of 2D crystals are available for these proteins. We have used artificially designed Zn(II) metal-ion binding sites to probe 7TM receptors structurally and functionally and to define some basic distance constraints for molecular modeling. In this way, the relative helical rotation and vertical translocation of transmembrane helices TM-II, TM-III, TM-V, and TM-VI of the tachykinin NK-1 receptor have been restricted. Collectively, these zinc sites constitute a basic network of distance constraints that limit the degrees of freedom of the interhelical contact faces in molecular models of 7TM receptors. The construction of artificially designed metal-ion sites is discussed also in the context of probes for conformational changes occurring during receptor activation.

Rational Design of Interleukin-21 Antagonist through Selective Elimination of the γC Binding Epitope

The cytokine interleukin (IL)-21 exerts pleiotropic effects acting through innate as well as adaptive immune responses. The activities of IL-21 are mediated through binding to its cognate receptor complex composed of the IL-21 receptor private chain (IL-21Rα) and the common γ-chain (γC), the latter being shared by IL-2, IL-4, IL-7, IL-9, and IL-15. The binding energy of the IL-21 ternary complex is predominantly provided by the high affinity interaction between IL-21 and IL-21Rα, whereas the interaction between IL-21 and γC, albeit essential for signaling, is rather weak. The design of IL-21 analogues, which have lost most or all affinity toward the signaling γC chain, while simultaneously maintaining a tight interaction with the private chain, would in theory represent candidates for IL-21 antagonists. We predicted the IL-21 residues, which compose the γC binding epitope using homology modeling and alignment with the related cytokines, IL-2 and IL-4. Next we systematically analyzed the predicted binding epitope by a mutagenesis study. Indeed two mutants, which have significantly impaired γC affinity with undiminished IL-21Rα affinity, were successfully identified. Functional studies confirmed that these two novel hIL-21 double mutants do act as hIL-21 antagonists.

How Receptor Mutagenesis May Confirm or Confuse Receptor Classification

To the knowledge of the authors there is no good example where mutagenesis has directly aided receptor classification as such. However, receptor mutagenesis has been used extensively to map receptor domains and single residues involved in ligand binding and G-protein coupling, which are important cornerstones in receptor classification. On the other hand, it could be argued that receptor mutagenesis in several cases has caused more confusion than clarification in this field. For example, some peptide systems have refused to present an agonist binding site which corresponds to the monoamine agonist binding site, and in some of these systems it has not been possible to obtain a nice overlap between binding sites for what is believed to be competitive ligands. Thus, either the mutational approachwhich is in no way flawless-is at fault in these cases, or some of our current dogmas of receptor activation and inactivation are not correct. In the following these issues will be addressed from a 7TM receptor point of view and with a bias for mutational analysis applied to characterizations of ligand binding sites.

Receptor structure-based discovery of non-metabolite agonists for the succinate receptor GPR91.

Objective Besides functioning as an intracellular metabolite, succinate acts as a stress-induced extracellular signal through activation of GPR91 (SUCNR1) for which we lack suitable pharmacological tools. Methods and results Here we first determined that the cis conformation of the succinate backbone is preferred and that certain backbone modifications are allowed for GPR91 activation. Through receptor modeling over the X-ray structure of the closely related P2Y1 receptor, we discovered that the binding pocket is partly occupied by a segment of an extracellular loop and that succinate therefore binds in a very different mode than generally believed. Importantly, an empty side-pocket is identified next to the succinate binding site. All this information formed the basis for a substructure-based search query, which, combined with molecular docking, was used in virtual screening of the ZINC database to pick two serial mini-libraries of a total of only 245 compounds from which sub-micromolar, selective GPR91 agonists of unique structures were identified. The best compounds were backbone-modified succinate analogs in which an amide-linked hydrophobic moiety docked into the side-pocket next to succinate as shown by both loss- and gain-of-function mutagenesis. These compounds displayed GPR91-dependent activity in altering cytokine expression in human M2 macrophages similar to succinate, and importantly were devoid of any effect on the major intracellular target, succinate dehydrogenase. Conclusions These novel, synthetic non-metabolite GPR91 agonists will be valuable both as pharmacological tools to delineate the GPR91-mediated functions of succinate and as leads for the development of GPR91-targeted drugs to potentially treat low grade metabolic inflammation and diabetic complications such as retinopathy and nephropathy.