The importance of interactions with helix 5 in determining the efficacy of β-adrenoceptor ligands

2013 ◽  
Vol 41 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Tony Warne ◽  
Christopher G. Tate
Keyword(s):  
Transmembrane Helix ◽  
Binding Pocket ◽  
Pharmacological Properties ◽  
Agonist Binding ◽  
Inverse Agonists ◽  
Inactive State ◽  
Partial Agonists ◽  
Activated State

Structures of the inactive state of the thermostabilized β1-adrenoceptor have been determined bound to eight different ligands, including full agonists, partial agonists, inverse agonists and biased agonists. Comparison of the structures shows distinct differences within the binding pocket that correlate with the pharmacological properties of the ligands. These data suggest that full agonists stabilize a structure with a contracted binding pocket and a rotamer change of serine (5.46) compared with when antagonists are bound. Inverse agonists may prevent both of these occurrences, whereas partial agonists stabilize a contraction of the binding pocket but not the rotamer change of serine (5.46). It is likely that subtle changes in the interactions between transmembrane helix 5 (H5) and H3/H4 on agonist binding promote the formation of the activated state.

scholarly journals Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

2007 ◽  
Vol 402 (1) ◽  
pp. 51-61 ◽  
Author(s):  
Brian J. Holleran ◽  
Marie-Eve Beaulieu ◽  
Christophe D. Proulx ◽  
Pierre Lavigne ◽  
Emanuel Escher ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Transmembrane Domain ◽  
Partial Agonist ◽  
Binding Pocket ◽  
Activation Mechanism ◽  
Urotensin Ii ◽  
Agonist Binding ◽  
Partial Agonists

The mechanism by which GPCRs (G-protein-coupled receptors) undergo activation is believed to involve conformational changes following agonist binding. We have used photoaffinity labelling to identify domains within GPCRs that make contact with various photoreactive ligands in order to better understand the activation mechanism. Here, a series of four agonist {[Bpa1]U-II (Bpa is p-benzoyl-L-phenylalanine), [Bpa2]U-II, [Bpa3]U-II and [Bpa4]U-II} and three partial agonist {[Bpa1Pen5D-Trp7Orn8]U-II (Pen is penicillamine), [Bpa2Pen5D-Trp7Orn8]U-II and [Pen5Bpa6D-Trp7Orn8]U-II} photoreactive urotensin II (U-II) analogues were used to identify ligand-binding sites on the UT receptor (U-II receptor). All peptides bound the UT receptor expressed in COS-7 cells with high affinity (Kd of 0.3–17.7 nM). Proteolytic mapping and mutational analysis led to the identification of Met288 of the third extracellular loop of the UT receptor as a binding site for all four agonist peptides. Both partial agonists containing the photoreactive group in positions 1 and 2 also cross-linked to Met288. We found that photolabelling with the partial agonist containing the photoreactive group in position 6 led to the detection of transmembrane domain 5 as a binding site for that ligand. Interestingly, this differs from Met184/Met185 of the fourth transmembrane domain that had been identified previously as a contact site for the full agonist [Bpa6]U-II. These results enable us to better map the binding pocket of the UT receptor. Moreover, the data also suggest that, although structurally related agonists or partial agonists may dock in the same general binding pocket, conformational changes induced by various states of activation may result in slight differences in spatial proximity within the cyclic portion of U-II analogues.

scholarly journals Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists

2020 ◽  
Vol 117 (46) ◽  
pp. 29144-29154
Author(s):  
Ali Işbilir ◽  
Jan Möller ◽  
Marta Arimont ◽  
Vladimir Bobkov ◽  
Cristina Perpiñá-Viciano ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transmembrane Helix ◽  
Binding Pocket ◽  
Cell Trafficking ◽  
Constitutive Activity ◽  
Inverse Agonists ◽  
Basal Activity ◽  
Cxc Chemokine Receptor 4 ◽  
Functional Relevance ◽  
Chemokine Receptor 4

Although class A G protein−coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer−dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

scholarly journals Why does Δ9-Tetrahydrocannabinol act as a partial agonist of cannabinoid receptors?

2021 ◽  
Author(s):  
Soumajit Dutta ◽  
Balaji Selvam ◽  
Aditi Das ◽  
Diwakar Shukla
Keyword(s):  
Side Effects ◽  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Partial Agonist ◽  
Transmembrane Helix ◽  
Toggle Switch ◽  
Agonist Binding ◽  
Partial Agonism ◽  
Partial Agonists ◽  
Agonist And Antagonist

AbstractCannabinoid receptor 1 (CB1) is a therapeutically relevant drug target for controlling pain, obesity, and other central nervous system disorders. However, full agonists and antagonists of CB1 have been reported to cause serious side effects in patients. Therefore, partial agonists have emerged as a viable alternative to full agonists and antagonists as they avoid overstimulation and side effects. One of the key bottlenecks in the design of partial agonists is the lack of understanding of the molecular mechanism of partial agonism. In this study, we examine two mechanistic hypotheses for the origin of partial agonism in cannabinoid receptors and explain the mechanistic basis of partial agonism exhibited by Δ9-Tetrahydrocannabinol (THC). In particular, we inspect whether partial agonism emerges from the ability of THC to bind in both agonist and antagonist binding pose or from its ability to only partially activate the receptor. Extensive molecular dynamics simulations and the Markov state model capture the THC binding in both antagonist, and agonist binding poses in CB1 receptor. Furthermore, we observe that binding of THC in the agonist binding pose leads to rotation of toggle switch residues and causes partial outward movement of intracellular transmembrane helix 6 (TM6). Our simulations also suggest that the alkyl side chain of THC plays a crucial role in determining partial agonism by stabilizing the ligand in the agonist and antagonist-like poses within the pocket. This study provides us fundamental insights into the mechanistic origin of the partial agonism of THC.

scholarly journals The endoplasmic reticulum P5A-ATPase is a transmembrane helix dislocase

Science ◽  
2020 ◽  
Vol 369 (6511) ◽  
pp. eabc5809 ◽  
Author(s):  
Michael J. McKenna ◽  
Sue Im Sim ◽  
Alban Ordureau ◽  
Lianjie Wei ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Targeting ◽  
Transmembrane Helix ◽  
Protein Composition ◽  
Binding Pocket ◽  
Transmembrane Segment ◽  
Substrate Binding Pocket ◽  
Cryo Electron Microscopy ◽  
Endogenous Substrate ◽  
P Type

Organelle identity depends on protein composition. How mistargeted proteins are selectively recognized and removed from organelles is incompletely understood. Here, we found that the orphan P5A–adenosine triphosphatase (ATPase) transporter ATP13A1 (Spf1 in yeast) directly interacted with the transmembrane segment (TM) of mitochondrial tail–anchored proteins. P5A-ATPase activity mediated the extraction of mistargeted proteins from the endoplasmic reticulum (ER). Cryo–electron microscopy structures of Saccharomyces cerevisiae Spf1 revealed a large, membrane-accessible substrate-binding pocket that alternately faced the ER lumen and cytosol and an endogenous substrate resembling an α-helical TM. Our results indicate that the P5A-ATPase could dislocate misinserted hydrophobic helices flanked by short basic segments from the ER. TM dislocation by the P5A-ATPase establishes an additional class of P-type ATPase substrates and may correct mistakes in protein targeting or topogenesis.

scholarly journals Lipid Bilayer Simulations of CXCR4 with Inverse Agonists and Weak Partial Agonists

2003 ◽  
Vol 278 (47) ◽  
pp. 47136-47144 ◽  
Author(s):  
John O. Trent ◽  
Zi-xuan Wang ◽  
James L. Murray ◽  
Wenhai Shao ◽  
Hirokazu Tamamura ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Inverse Agonists ◽  
Partial Agonists

scholarly journals Transmembrane signaling and cytoplasmic signal conversion by dimeric transmembrane helix 2 and a linker domain of the DcuS sensor kinase

2020 ◽  
pp. jbc.RA120.015999
Author(s):  
Marius Stopp ◽  
Philipp Aloysius Steinmetz ◽  
Christopher Schubert ◽  
Christian Griesinger ◽  
Dirk Schneider ◽  
...  
Keyword(s):  
Signal Transmission ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Kinase Domain ◽  
Sensor Kinase ◽  
Transmembrane Signaling ◽  
Activated State ◽  
Sensor Kinases ◽  
And Function ◽  
Cytoplasmic Side

Transmembrane signaling is a key process of membrane bound sensor kinases. The C4-dicarboxylate (fumarate) responsive sensor kinase DcuS of Escherichia coli is anchored by transmembrane helices TM1 and TM2 in the membrane. Signal transmission across the membrane relies on the piston-type movement of the periplasmic part of TM2. To define the role of TM2 in transmembrane signaling, we use oxidative Cys cross-linking to demonstrate that TM2 extends over the full distance of the membrane and forms a stable transmembrane homodimer in both the inactive and fumarate-activated state of DcuS. A S186xxxGxxxG194 motif is required for the stability and function of the TM2 homodimer. The TM2 helix further extends on the periplasmic side into the α6-helix of the sensory PASP domain, and on the cytoplasmic side into the α1-helix of PASC. PASC has to transmit the signal to the C-terminal kinase domain. A helical linker on the cytoplasmic side connecting TM2 with PASC contains a LxxxLxxxL sequence. The dimeric state of the linker was relieved during fumarate activation of DcuS, indicating structural rearrangements in the linker. Thus, DcuS contains a long α-helical structure reaching from the sensory PASP (α6) domain across the membrane to α1(PASC). Taken together, the results suggest piston-type transmembrane signaling by the TM2-homodimer from PASP across the full TM region, whereas the fumarate-destabilized linker dimer converts the signal on the cytoplasmic side for PASC and kinase regulation.

scholarly journals Analysis of full and partial agonists binding to β2-adrenergic receptor suggests a role of transmembrane helix V in agonist-specific conformational changes

2009 ◽  
Vol 22 (4) ◽  
pp. 307-318 ◽  
Author(s):  
Vsevolod Katritch ◽  
Kimberly A. Reynolds ◽  
Vadim Cherezov ◽  
Michael A. Hanson ◽  
Christopher B. Roth ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Adrenergic Receptor ◽  
Transmembrane Helix ◽  
Partial Agonists ◽  
Β2 Adrenergic Receptor

2-[N-Acylamino(C1−C3)alkyl]indoles as MT1Melatonin Receptor Partial Agonists, Antagonists, and Putative Inverse Agonists

1998 ◽  
Vol 41 (19) ◽  
pp. 3624-3634 ◽  
Author(s):  
Gilberto Spadoni ◽  
Cesarino Balsamini ◽  
Annalida Bedini ◽  
Giuseppe Diamantini ◽  
Barbara Di Giacomo ◽  
...  
Keyword(s):  
Inverse Agonists ◽  
Partial Agonists

scholarly journals Mapping the ρ1GABACReceptor Agonist Binding Pocket

2004 ◽  
Vol 280 (2) ◽  
pp. 1535-1542 ◽  
Author(s):  
Anna Sedelnikova ◽  
Craig D. Smith ◽  
Stanislav O. Zakharkin ◽  
Delores Davis ◽  
David S. Weiss ◽  
...  
Keyword(s):  
Binding Pocket ◽  
Agonist Binding

scholarly journals CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state.

1996 ◽  
Vol 107 (1) ◽  
pp. 103-119 ◽  
Author(s):  
D J Wilkinson ◽  
M K Mansoura ◽  
P Y Watson ◽  
L S Smit ◽  
F S Collins ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Atp Hydrolysis ◽  
Nucleotide Binding ◽  
Binding Pocket ◽  
Active State ◽  
Slow Phase ◽  
Atp Binding ◽  
Wild Type ◽  
Rate Analysis ◽  
Activated State

The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state. These results are consistent with a hypothesis for CFTR activation that invokes the binding and hydrolysis of ATP at NBF1 as a crucial step in activation, while at NBF2, ATP binding enhances access to the active state, but the rate of ATP hydrolysis controls the duration of the active state. The relatively slow time courses for activation and deactivation suggest that slow processes modulate ATP-dependent gating.

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