Rie Nygaard

A Conserved Aromatic Lock for the Tryptophan Rotameric Switch in TM-VI of Seven-transmembrane Receptors

The conserved tryptophan in position 13 of TM-VI (Trp-VI:13 or Trp-6.48) of the CWXP motif located at the bottom of the main ligand-binding pocket in TM-VI is believed to function as a rotameric microswitch in the activation process of seven-transmembrane (7TM) receptors. Molecular dynamics simulations in rhodopsin demonstrated that rotation around the chi1 torsion angle of Trp-VI:13 brings its side chain close to the equally highly conserved Phe-V:13 (Phe-5.47) in TM-V. In the ghrelin receptor, engineering of high affinity metal-ion sites between these positions confirmed their close spatial proximity. Mutational analysis was performed in the ghrelin receptor with multiple substitutions and with Ala substitutions in GPR119, GPR39, and the β2-adrenergic receptor as well as the NK1 receptor. In all of these cases, it was found that mutation of the Trp-VI:13 rotameric switch itself eliminated the constitutive signaling and strongly impaired agonist-induced signaling without affecting agonist affinity and potency. Ala substitution of Phe-V:13, the presumed interaction partner for Trp-VI:13, also in all cases impaired both the constitutive and the agonist-induced receptor signaling, but not to the same degree as observed in the constructs where Trp-VI:13 itself was mutated, but again without affecting agonist potency. In a proposed active receptor conformation generated by molecular simulations, where the extracellular segment of TM-VI is tilted inwards in the main ligand-binding pocket, Trp-VI:13 could rotate into a position where it obtained an ideal aromatic-aromatic interaction with Phe-V:13. It is concluded that Phe-V:13 can serve as an aromatic lock for the proposed active conformation of the Trp-VI:13 rotameric switch, being involved in the global movement of TM-V and TM-VI in 7TM receptor activation.

Identification of an Efficacy Switch Region in the Ghrelin Receptor Responsible for Interchange between Agonism and Inverse Agonism

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this “efficacy shift region” on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation.

Conserved Water-mediated Hydrogen Bond Network between TM-I, -II, -VI, and -VII in 7TM Receptor Activation

Five highly conserved polar residues connected by a number of structural water molecules together with two rotamer micro-switches, TrpVI:13 and TyrVII:20, constitute an extended hydrogen bond network between the intracellular segments of TM-I, -II, -VI, and -VII of 7TM receptors. Molecular dynamics simulations showed that, although the fewer water molecules in rhodopsin were relatively movable, the hydrogen bond network of the β2-adrenergic receptor was fully loaded with water molecules that were surprisingly immobilized between the two rotamer switches, both apparently being in their closed conformation. Manipulations of the rotamer state of TyrVII:20 and TrpVI:13 demonstrated that these residues served as gates for the water molecules at the intracellular and extracellular ends of the hydrogen bond network, respectively. TrpVI:13 at the bottom of the main ligand-binding pocket was shown to apparently function as a catching trap for water molecules. Mutational analysis of the β2-adrenergic receptor demonstrated that the highly conserved polar residues of the hydrogen bond network were all important for receptor signaling but served different functions, some dampening constitutive activity (AsnI:18, AspII:10, and AsnVII:13), whereas others (AsnVII:12 and AsnVII:16) located one helical turn apart and sharing a water molecule were shown to be essential for agonist-induced signaling. It is concluded that the conserved water hydrogen bond network of 7TM receptors constitutes an extended allosteric interface between the transmembrane segments being of crucial importance for receptor signaling and that part of the function of the rotamer micro-switches, TyrVII:20 and TrpVI:13, is to gate or trap the water molecules.

Activation of the CXCR3 chemokine receptor through anchoring of a small molecule chelator ligand between TM-III, -IV and -VI

Seven transmembrane segment (7TM) receptors are activated through a common, still rather unclear molecular mechanism by a variety of chemical messengers ranging from monoamines to large proteins. By introducing a His residue at position III:05 in the CXCR3 receptor a metal ion site was built between the extracellular ends of transmembrane (TM) III and TM-IV to anchor aromatic chelators at a location corresponding to the presumed binding pocket for adrenergic receptor agonists. In this construct, free metal ions had no agonistic effect in accordance with the optimal geometry of the metal ion site in molecular models built over the inactive form of rhodopsin. In contrast, the aromatic chelators bipyridine or phenanthrolene in complex with Zn(II) or Cu(II) acted as potent agonists displaying signaling efficacies similar to or even better than the endogenous chemokine agonists. Molecular modeling and molecular simulations combined with mutational analysis indicated that the metal ion site-anchored chelators act as agonists by establishing an aromatic-aromatic, second-site interaction with TyrVI:16 on the inner face of TM-VI. It is noteworthy that this interaction required that the extracellular segment of TM-VI moves inward in the direction of TM-III, whereby TyrVI:16 together with the chelators complete an “aromatic zipper” also comprising PheIII:08 (corresponding to the monoamine receptor anchoring point) and TyrVII:10 (corresponding to the retinal attachment site in rhodopsin). Chemokine agonism was independent of this aromatic zipper. It is proposed that in rhodopsin-like 7TM receptors, small-molecule compounds in general act as agonists in a similar manner as here demonstrated with the artificial, metal ion site anchored chelators, by holding TM-VI bent inward.

Molecular Interaction of a Potent Nonpeptide Agonist with the Chemokine Receptor CCR8

Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spiro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation, and calcium release with high potencies (EC50 from 11 to 87 nM) and with efficacies similar to that of the endogenous agonist CCL1, and it competed for 125I-CCL1 binding with an affinity of 66 nM. A series of 29 mutations targeting 25 amino acids broadly distributed in the minor and major ligand-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hydroxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1yl)butanamide (LMD-268), and N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-1,2,3,4-tetrahydro-2-oxoquinoline-4-carboxamide (LMD-174)] included several key-residues for nonpeptide antagonists targeting CCR1, -2, and -5. It is noteworthy that a decrease in potency of nearly 1000-fold was observed for all five compounds for the Ala substitution of the anchor-point GluVII:06 (Glu(286)) and a gain-of-function of 19-fold was observed for LMD-009 (but not the four other analogs) for the Ala substitution of PheVI:16 (Phe(254)). These structural hallmarks were particularly important in the generation of a model of the molecular mechanism of action for LMD-009. In conclusion, we present the first molecular mapping of the interaction of a nonpeptide agonist with a chemokine receptor and show that the binding pocket of LMD-009 and of analogs overlaps considerably with the binding pockets of CC-chemokine receptor nonpeptide antagonists in general.

The Arginine of the DRY Motif in Transmembrane Segment III Functions as a Balancing Micro-switch in the Activation of the β2-Adrenergic Receptor

Background: ArgIII:26 (Arg3.50) is almost 100% conserved among 7TM receptors. Results: Alanine substitution of ArgIII:26 was basically silent while providing expected positive and negative effects when applied to its proposed interaction partners. Conclusion: The ArgIII:26 micro-switch stabilizes both active and inactive receptor conformations but is not directly involved in receptor signaling. Significance: This work provides clarification of the molecular mechanism of a crucial micro-switch in 7TM receptors. Recent high resolution x-ray structures of the β2-adrenergic receptor confirmed a close salt-bridge interaction between the suspected micro-switch residue ArgIII:26 (Arg3.50) and the neighboring AspIII:25 (Asp3.49). However, neither the expected “ionic lock” interactions between ArgIII:26 and GluVI:-06 (Glu6.30) in the inactive conformation nor the interaction with TyrV:24 (Tyr5.58) in the active conformation were observed in the x-ray structures. Here we find through molecular dynamics simulations, after removal of the stabilizing T4 lysozyme, that the expected salt bridge between ArgIII:26 and GluVI:-06 does form relatively easily in the inactive receptor conformation. Moreover, mutational analysis of GluVI:-06 in TM-VI and the neighboring AspIII:25 in TM-III demonstrated that these two residues do function as locks for the inactive receptor conformation as we observed increased Gs signaling, arrestin mobilization, and internalization upon alanine substitutions. Conversely, TyrV:24 appears to play a role in stabilizing the active receptor conformation as loss of function of Gs signaling, arrestin mobilization, and receptor internalization was observed upon alanine substitution of TyrV:24. The loss of function of the TyrV:24 mutant could partly be rescued by alanine substitution of either AspIII:25 or GluVI:-06 in the double mutants. Surprisingly, removal of the side chain of the ArgIII:26 micro-switch itself had no effect on Gs signaling and internalization and only reduced arrestin mobilization slightly. It is suggested that ArgIII:26 is equally important for stabilizing the inactive and the active conformation through interaction with key residues in TM-III, -V, and -VI, but that the ArgIII:26 micro-switch residue itself apparently is not essential for the actual G protein activation.

Model-Based Discovery of Synthetic Agonists for the Zn2+-Sensing G-Protein-Coupled Receptor 39 (GPR39) Reveals Novel Biological Functions

The G-protein-coupled receptor 39 (GPR39) is a G-protein-coupled receptor activated by Zn2+. We used a homology model-based approach to identify small-molecule pharmacological tool compounds for the receptor. The method focused on a putative binding site in GPR39 for synthetic ligands and knowledge of ligand binding to other receptors with similar binding pockets to select iterative series of minilibraries. These libraries were cherry-picked from all commercially available synthetic compounds. A total of only 520 compounds were tested in vitro, making this method broadly applicable for tool compound development. The compounds of the initial library were inactive when tested alone, but lead compounds were identified using Zn2+ as an allosteric enhancer. Highly selective, highly potent Zn2+-independent GPR39 agonists were found in subsequent minilibraries. These agonists identified GPR39 as a novel regulator of gastric somatostatin secretion.

Injuries during football tournaments in 45,000 children and adolescents

Abstract Four percent of the world’s population, or 265 million people, play football, and many players are injured every year. The present study investigated more than 1800 injuries in over 45,000 youth players participating in three consecutive international football tournaments in Denmark in 2012–2014. The aim was to investigate the injury types and locations in children and adolescent football players and the differences between genders and age groups (11–15 and 16–19 years of age). An overall injury rate of 15.3 per 1000 player hours was found. The most common injury location was lower extremities (66.7%), and the most common injury type was contusion (24.4%). Girls had a relative risk of injury of 1.5 compared with boys, p < .001, and they had a higher proportion of injuries to knee and lower leg, 23.8%, than boys, 19.0%, p < .01. Boys had a higher proportion of fracture, 6.8%, as opposed to 3.3% among girls, p < .001. In conclusion, we found the youngest girls to have a higher incidence of almost all injury categories than any other group. In general, the incidence of injury decreased with age. The study provides a detailed insight into the injuries that may be expected at a large youth football tournament. These findings are of great value for organizations and healthcare professionals planning similar events and for planning injury prevention strategies, which would be of special interest in the youngest female players in general.

Response to resistance training following immobilization-Influence of delaying post-exercise meal

Elderly women are more vulnerable to muscle disuse atrophy than men, due to lower muscle mass. Thus, identification of preventive strategies is of principal interest. We studied effects of training and timing of protein‐rich meals after exercise sessions, on skeletal muscle mass in healthy postmenopausal women. Following 2 weeks of one‐legged immobilization, a protein‐rich meal was ingested either immediately (T0; n = 9, 56 ± 2 years) or 2 hours (T2; n = 8, 57 ± 3 years) after termination of resistance exercise sessions for 6 weeks. Muscle mass, strength, and power were assessed before, after 2 weeks of lower limb immobilization and after 2 and 6 weeks of combined resistance training and diet intervention. Immobilization reduced muscle mass (~10%), strength (~23%), and power (~26%), but was returned to baseline levels after 2 weeks of training. Improvements in muscle mass and strength did not differ between groups. T0, but not T2, increased their total daily protein intake during the training period. In conclusion, only 2 weeks of supervised resistance training counteracted reductions in muscle mass and strength in postmenopausal women after 2 weeks of immobilization. Timing of protein‐rich meals in the hours after resistance exercise did not have any impact on the effects of training.

Biomechanical properties of the patellar tendon in children with heritable connective tissue disorders

PurposeHereditary connective tissue disorders (HCTDs), such as classic Ehlers–Danlos syndrome (cEDS) and Marfan syndrome (MS) share overlapping features like hypermobility and tissue fragility. In clinical practice it remains a challenge to distinguish children and adolescents with HCTD from healthy children. The purpose of this study was to investigate the biomechanical properties of the patellar tendon and joint laxity (Beighton score) in children with HCTDs (n = 7) compared to healthy controls (n = 14).MethodsThe mechanical properties of the patellar tendon were assessed using simultaneous force and ultrasonographic measurements during isometric ramp contractions. Ultrasonography was also used to measure tendon dimensions. The HCTD children were matched with 2 healthy controls with regard to age, body mass index (BMI), sex and physical activity level.ResultsThe HCTD children had a greater degree of joint laxity (P < 0.01). Although, the patellar tendon dimensions did not differ significantly between the two groups, the HCTD children showed a tendency toward a larger patellar tendon cross-sectional area (CSA) (35%, P = 0.19). Moreover, stiffness did not differ between the two groups, but secant modulus was 27% lower in children with a HCTD (P = 0.05) at common force and 34% lower at maximum force (P = 0.02).ConclusionsThe present study demonstrates for the first time that children with HCTDs have lower material properties (modulus) of their patellar tendon, which may be indicative of general impairment of connective tissue mechanics related to their increased joint laxity.