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

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.

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Fluorometric study on conformational changes in the catalytic cycle of sarcoplasmic reticulum Ca2+-ATPase

Bioscience Reports ◽

10.1007/bf01788364 ◽

1995 ◽

Vol 15 (5) ◽

pp. 317-326 ◽

Cited By ~ 4

Author(s):

Tohru Kanazawa ◽

Hiroshi Suzuki ◽

Takashi Daiho ◽

Kazuo Yamasaki

Keyword(s):

Sarcoplasmic Reticulum ◽

Binding Site ◽

Conformational Changes ◽

Binding Sites ◽

Transmembrane Domain ◽

Tryptophan Fluorescence ◽

Cytoplasmic Domain ◽

Catalytic Cycle ◽

Atp Binding ◽

Atp Binding Site

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps; conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps.

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Positive co-operative binding at two weak lysine-binding sites governs the Glu-plasminogen conformational change

Biochemical Journal ◽

10.1042/bj2850419 ◽

1992 ◽

Vol 285 (2) ◽

pp. 419-425 ◽

Cited By ~ 34

Author(s):

U Christensen ◽

L Mølgaard

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Change ◽

Conformational Changes ◽

Binding Sites ◽

High Specificity ◽

Stopped Flow ◽

Protein Fluorescence ◽

Lysine Residues ◽

Kinetics Of

The kinetics of a series of Glu-plasminogen ligand-binding processes were investigated at pH 7.8 and 25 degrees C (in 0.1 M-NaCl). The ligands include compounds analogous to C-terminal lysine residues and to normal lysine residues. Changes of the Glu-plasminogen protein fluorescence were measured in a stopped-flow instrument as a function of time after rapid mixing of Glu-plasminogen and ligand at various concentrations. Large positive fluorescence changes (approximately 10%) accompany the ligand-induced conformational changes of Glu-plasminogen resulting from binding at weak lysine-binding sites. Detailed studies of the concentration-dependencies of the equilibrium signals and the rate constants of the process induced by various ligands showed the conformational change to involve two sites in a concerted positive co-operative process with three steps: (i) binding of a ligand at a very weak lysine-binding site that preferentially, but not exclusively, binds C-terminal-type lysine ligands, (ii) the rate-determining actual-conformational-change step and (iii) binding of one more lysine ligand at a second weak lysine-binding site that then binds the ligand more tightly. Further, totally independent initial small negative fluorescence changes (approximately 2-4%) corresponding to binding at the strong lysine-binding site of kringle 1 [Sottrup-Jensen, Claeys, Zajdel, Petersen & Magnusson (1978) Prog. Chem. Fibrinolysis Thrombolysis 3, 191-209] were observed for the C-terminal-type ligands. The finding that the conformational change in Glu-plasminogen involves two weak lysine-binding sites indicates that the effect cannot be assigned to any single kringle and that the problem of whether kringle 4 or kringle 5 is responsible for the process resolves itself. Probably kringle 4 and 5 are both participating. The involvement of two lysine binding-sites further makes the high specificity of Glu-plasminogen effectors more conceivable.

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String method solution of the gating pathways for a pentameric ligand-gated ion channel

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617567114 ◽

2017 ◽

Vol 114 (21) ◽

pp. E4158-E4167 ◽

Cited By ~ 27

Author(s):

Bogdan Lev ◽

Samuel Murail ◽

Frédéric Poitevin ◽

Brett A. Cromer ◽

Marc Baaden ◽

...

Keyword(s):

Free Energy ◽

Conformational Changes ◽

Transmembrane Domain ◽

Minimum Free Energy ◽

Agonist Binding ◽

String Method ◽

Transition Analysis ◽

Subunit Interface ◽

Ion Conducting ◽

Allosteric Mechanisms

Pentameric ligand-gated ion channels control synaptic neurotransmission by converting chemical signals into electrical signals. Agonist binding leads to rapid signal transduction via an allosteric mechanism, where global protein conformational changes open a pore across the nerve cell membrane. We use all-atom molecular dynamics with a swarm-based string method to solve for the minimum free-energy gating pathways of the proton-activated bacterial GLIC channel. We describe stable wetted/open and dewetted/closed states, and uncover conformational changes in the agonist-binding extracellular domain, ion-conducting transmembrane domain, and gating interface that control communication between these domains. Transition analysis is used to compute free-energy surfaces that suggest allosteric pathways; stabilization with pH; and intermediates, including states that facilitate channel closing in the presence of an agonist. We describe a switching mechanism that senses proton binding by marked reorganization of subunit interface, altering the packing of β-sheets to induce changes that lead to asynchronous pore-lining M2 helix movements. These results provide molecular details of GLIC gating and insight into the allosteric mechanisms for the superfamily of pentameric ligand-gated channels.

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Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

International Journal of Molecular Sciences ◽

10.3390/ijms20061444 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1444 ◽

Cited By ~ 4

Author(s):

Soria Iatmanen-Harbi ◽

lucile Senicourt ◽

Vassilios Papadopoulos ◽

Olivier Lequin ◽

Jean-Jacques Lacapere

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Conformational Changes ◽

Protoporphyrin Ix ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Translocator Protein ◽

Binding Affinities ◽

Ligand Selectivity ◽

High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

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One Intact Transmembrane Substrate Binding Site Is Sufficient for the Function of the Homodimeric Type I ATP-Binding Cassette Importer for Positively Charged Amino Acids Art(MP) 2 of Geobacillus stearothermophilus

Journal of Bacteriology ◽

10.1128/jb.00092-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 2

Author(s):

Johanna Heuveling ◽

Heidi Landmesser ◽

Erwin Schneider

Keyword(s):

Binding Site ◽

Binding Sites ◽

Substrate Binding ◽

Binding Pocket ◽

Atp Binding ◽

Content Type ◽

Charged Amino Acids ◽

Substrate Binding Sites ◽

Positively Charged ◽

Atp Binding Cassette

ABSTRACT ATP-binding cassette (ABC) transport systems comprise two transmembrane domains/subunits that form a translocation path and two nucleotide-binding domains/subunits that bind and hydrolyze ATP. Prokaryotic canonical ABC import systems require an extracellular substrate-binding protein for function. Knowledge of substrate-binding sites within the transmembrane subunits is scarce. Recent crystal structures of the ABC importer Art(QN) 2 for positively charged amino acids of Thermoanerobacter tengcongensis revealed the presence of one substrate molecule in a defined binding pocket in each of the transmembrane subunits, ArtQ (J. Yu, J. Ge, J. Heuveling, E. Schneider, and M. Yang, Proc Natl Acad Sci U S A 112:5243–5248, 2015, https://doi.org/10.1073/pnas.1415037112 ). This finding raised the question of whether both sites must be loaded with substrate prior to initiation of the transport cycle. To address this matter, we first explored the role of key residues that form the binding pocket in the closely related Art(MP) 2 transporter of Geobacillus stearothermophilus , by monitoring consequences of mutations in ArtM on ATPase and transport activity at the level of purified proteins embedded in liposomes. Our results emphasize that two negatively charged residues (E153 and D160) are crucial for wild-type function. Furthermore, the variant Art[M(L67D)P] 2 exhibited strongly impaired activities, which is why it was considered for construction of a hybrid complex containing one intact and one impaired substrate-binding site. Activity assays clearly revealed that one intact binding site was sufficient for function. To our knowledge, our study provides the first biochemical evidence on transmembrane substrate-binding sites of an ABC importer. IMPORTANCE Canonical prokaryotic ATP-binding cassette importers mediate the uptake of a large variety of chemicals, including nutrients, osmoprotectants, growth factors, and trace elements. Some also play a role in bacterial pathogenesis, which is why full understanding of their mode of action is of the utmost importance. One of the unsolved problems refers to the chemical nature and number of substrate binding sites formed by the transmembrane subunits. Here, we report that a hybrid amino acid transporter of G. stearothermophilus , encompassing one intact and one impaired transmembrane binding site, is fully competent in transport, suggesting that the binding of one substrate molecule is sufficient to trigger the translocation process.

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Synthetic site-directed ligands

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1989.0027 ◽

1989 ◽

Vol 323 (1217) ◽

pp. 495-509 ◽

Cited By ~ 14

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Opioid Peptides ◽

Binding Pocket ◽

Tyrosine Residues ◽

Chemotactic Peptides ◽

Binding Cavity ◽

False Floor ◽

Type Site ◽

Main Cavity

Complexes of nucleotides, peptides and arom atic hapten-like compounds with immunoglobulin fragments were studied by X-ray analysis. Alter tri- or hexanucleotides of deoxythymidylate were diffused into triclinic crystals of a Fab (BV04- 01) with specificity for single-stranded DNA, extensive changes were detected throughout the structure of the protein. The Fab co-crystallized with a tri- or pentanucleotide in a different space group (monoclinic), an observation sometimes correlated with alterations in the structure of the ‘native’ protein. Structural analyses of the co-crystals are in progress for direct comparisons with the unliganded Fab. In crystals of a human (Meg) Bence-Jones dimer, synthetic opioid peptides, chemotactic peptides or dinitrophenyl (DNP) derivatives could be diffused into a large conical binding cavity. The conformations of both the ligand and the protein were usually altered during the binding process. At the base of the cavity tyrosine residues could be displaced like trap-doors to permit entry of some opioid peptides and DNP compounds into a deep binding pocket. In co-crystals of the dimer and bis(DNP)lysine, two ligand molecules were bound in tandem, one in the main cavity and the second in the deep pocket. One ligand adopted an extended conformation, with the ε-DNP ring near the floor of the main cavity and the α-DNP group in solvent outside the binding site. There were no significant conformational changes in the protein. In contrast, the second ligand was very compact, with both DNP rings immersed in the deep pocket, and the binding site was expanded to accommodate the oversized ligand. Peptides designed to be specific for the main cavity were incrementally constructed from minimal binding units by M. Geysen, G. Trippick, S. Rodda and their colleagues. A pentapeptide optimized for binding by this method was diffused into a crystal of the dimer and found by Fourier difference analysis to lodge exclusively in the main cavity as predicted. Binding regions in the BV04-01 Fab and the Meg dimer were markedly different in size and shape. The Fab had a groove-type site, in which a layer of sidechains acted like a false floor over regions analogous to the cavity and deep pocket of the Bence-Jones dimer.

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

Biochemical Society Transactions ◽

10.1042/bst20120228 ◽

2013 ◽

Vol 41 (1) ◽

pp. 159-165 ◽

Cited By ~ 18

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.

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Comparison of interactions of R-(+)- and S-(−)-isomers of β-adrenergic partial agonists, befunolol and carteolol, with high affinity site of β-adrenoceptors in isolated rabbit ciliary body and guinea-pig taenia caeci

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y91-144 ◽

1991 ◽

Vol 69 (7) ◽

pp. 951-957 ◽

Cited By ~ 6

Author(s):

Katsuo Koike ◽

Hisashi Hagiwara ◽

Issei Takayanagi

Keyword(s):

Cyclic Amp ◽

Guinea Pig ◽

Binding Site ◽

Ciliary Body ◽

Partial Agonist ◽

Affinity Binding ◽

High Affinity Binding ◽

High Affinity ◽

Partial Agonists ◽

High Affinity Binding Site

The stereoselectivities of β-adrenergic partial agonists for the high affinity binding site of β-adrenoceptors in the rabbit ciliary body and the guinea-pig taenia caeci were studied. The pA2 values of the S-(−)-isomers of befunolol and carteolol against S-(−)-isoprenaline, which were calculated from the shift of each concentration–response curve in increasing cyclic AMP levels, were significantly larger than those of the R-(+)-isomers in the guinea-pig taenia caeci, while the pA2 values of the S-(−)-isomers were not significantly larger than those of the R-(+)-isomers in the rabbit ciliary body. The pKi values determined from the binding experiments were in good agreement with the pA2 values from the increases in cyclic AMP levels. These results suggest that the high affinity binding site of β-adrenoceptors in the guinea-pig taenia caeci may be able to discriminate stereoselectively between the R-(+)- and S-(−)-isomers, while in the rabbit ciliary body there is no stereoselectivity between the two enantiomers.Key words: stereoselectivity, β-adrenoceptor, partial agonist, rabbit ciliary body, guinea-pig taenia caeci.

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Anti-diabetic drug binding site in KATP channels revealed by Cryo-EM

10.1101/172908 ◽

2017 ◽

Cited By ~ 2

Author(s):

Gregory M. Martin ◽

Balamurugan Kandasamy ◽

Frank DiMaio ◽

Craig Yoshioka ◽

Show-Ling Shyng

Keyword(s):

Binding Site ◽

Binding Sites ◽

Katp Channels ◽

Binding Pocket ◽

Drug Binding ◽

Channel Activity ◽

Nucleotide Binding Domain ◽

High Affinity ◽

Drug Binding Site ◽

First Time

AbstractSulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8Å resolution, which reveals in unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

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Self-association configures the NAD+-binding site of plant NLR TIR domains

10.1101/2021.10.02.462850 ◽

2021 ◽

Author(s):

Hayden Burdett ◽

Xiahao Hu ◽

Maxwell X Rank ◽

Natsumi Maruta ◽

Bostjan Kobe

Keyword(s):

Binding Site ◽

Conformational Changes ◽

Active Site ◽

Binding Sites ◽

Molecular Mechanisms ◽

Structural Features ◽

Active Conformation ◽

Effector Triggered Immunity ◽

Self Association ◽

Tir Domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

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