scholarly journals Molecular sampling of the allosteric binding pocket of the TSH receptor provides discriminative pharmacophores for antagonist and agonists

2013 ◽  
Vol 41 (1) ◽  
pp. 213-217 ◽  
Author(s):  
Inna Hoyer ◽  
Ann-Karin Haas ◽  
Annika Kreuchwig ◽  
Ralf Schülein ◽  
Gerd Krause
Keyword(s):  
Amino Acids ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Binding Pocket ◽  
Trigger Points ◽  
Antagonistic Effect ◽  
Site Directed Mutagenesis ◽  
Detailed Knowledge ◽  
Contact Points ◽  
Allosteric Binding Pocket

The TSHR (thyrotropin receptor) is activated endogenously by the large hormone thyrotropin and activated pathologically by auto-antibodies. Both activate and bind at the extracellular domain. Recently, SMLs (small-molecule ligands) have been identified, which bind in an allosteric binding pocket within the transmembrane domain. Modelling driven site-directed mutagenesis of amino acids lining this pocket led to the delineation of activation and inactivation sensitive residues. Modified residues showing CAMs (constitutively activating mutations) indicate signalling-sensitive positions and mark potential trigger points for agonists. Silencing mutations lead to an impairment of basal activity and mark contact points for antagonists. Mapping these residues on to a structural model of TSHR indicates locations where an SML may switch the receptor to an inactive or active conformation. In the present article, we report the effects of SMLs on these signalling-sensitive amino acids at the TSHR. Surprisingly, the antagonistic effect of SML compound 52 was reversed to an agonistic effect, when tested at the CAM Y667A. Switching agonism to antagonism and the reverse by changing either SMLs or residues covering the binding pocket provides detailed knowledge about discriminative pharmacophores. It prepares the basis for rational optimization of new high-affinity antagonists to interfere with the pathogenic activation of the TSHR.

scholarly journals Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor

2003 ◽  
Vol 371 (2) ◽  
pp. 443-449 ◽  
Author(s):  
Frank NEUSCHÄFER-RUBE ◽  
Eva ENGEMAIER ◽  
Sina KOCH ◽  
Ulrike BÖER ◽  
Gerhard P. PÜSCHEL
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
G Protein ◽  
Transmembrane Domain ◽  
Sequence Similarity ◽  
Membrane Receptors ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Receptor Subtypes ◽  
Plasma Membrane Receptors

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF2α and PGD2. Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF2α-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation.

scholarly journals Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

2010 ◽  
Vol 76 (5) ◽  
pp. 1653-1660 ◽  
Author(s):  
Ponnandy Prabhu ◽  
Marimuthu Jeya ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Licheniformis ◽  
Catalytic Efficiency ◽  
Homology Model ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
High Turnover ◽  
Arabinose Isomerase

ABSTRACT Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

scholarly journals Identification of a functionally critical GXXG motif and its relationship to the folate binding site of the proton-coupled folate transporter (PCFT-SLC46A1)

2012 ◽  
Vol 303 (6) ◽  
pp. C673-C681 ◽  
Author(s):  
Rongbao Zhao ◽  
Daniel Sanghoon Shin ◽  
Andras Fiser ◽  
I. David Goldman
Keyword(s):  
Functional Role ◽  
Autosomal Recessive ◽  
Structural Model ◽  
Transmembrane Domain ◽  
Autosomal Recessive Disorder ◽  
Binding Pocket ◽  
Loss Of Function ◽  
Substituted Cysteine Accessibility ◽  
Translocation Pathway

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption, and loss-of-function mutations in this gene result in the autosomal recessive disorder hereditary folate malabsorption. The current study, focused on a structure-functional analysis of this transporter, identified Gly-189 and Gly-192 (a GxxG motif) located in the fifth transmembrane domain as residues that could not be replaced with alanine without a loss of function. In contrast, function was preserved when Gly-56 and Gly-59 (the other conservative GXXG motif in human PCFT) were replaced with alanine. Similarly, Gly-93 and Gly-97, which constitute the only conserved GXXXG dimerization motif in human PCFT, tolerated alanine substitution. To explore the role of this region in folate binding, the residues around Gly-189 and Gly-192 were analyzed by the substituted cysteine accessibility method. Both I188C and M193C mutants were functional and were inhibited by membrane-impermeable sulfhydryl-reactive reagents; this could be prevented with PCFT substrate, but the protection was sustained at 0°C only for the I188C mutant, consistent with localization of Ile-188 in the PCFT folate binding pocket. The functional role of residues around Gly-189 and Gly-192 is consistent with a molecular structural model in which these two residues along with Ieu-188 are accessible to the PCFT aqueous translocation pathway.

scholarly journals Activation of GPR116/ADGRF5 by its tethered agonist requires key amino acids in extracellular loop 2 of the transmembrane domain

2021 ◽  
Author(s):  
James P Bridges ◽  
Caterina Safina ◽  
Bernard Picard ◽  
Kari Brown ◽  
Alyssa Filuta ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Peptide Sequence ◽  
Knock Out ◽  
Autocatalytic Cleavage ◽  
Knock Out Mice

The mechanistic details of the tethered agonist mode of activation for adhesion GPCRs has not been completely deciphered. We set out to investigate the physiologic importance of autocatalytic cleavage upstream of the agonistic peptide sequence, an event necessary for NTF displacement and subsequent receptor activation. To examine this hypothesis, we characterized tethered agonist-mediated activation of GPR116 in vitro and in vivo. A knock-in mouse expressing a non-cleavable GPR116 mutant phenocopies the pulmonary phenotype of GPR116 knock-out mice, demonstrating that tethered agonist-mediated receptor activation is indispensable for function in vivo. Using site-directed mutagenesis and species swapping approaches we identified key conserved amino acids for GPR116 activation in the tethered agonist sequence and in extracellular loops 2/3 (ECL2/3). We further highlight residues in transmembrane7 (TM7) that mediate stronger signaling in mouse versus human GPR116 and recapitulate these findings in a model supporting tethered agonist:ECL2 interactions for GPR116 activation.

scholarly journals The Vaccinia Virus 14-Kilodalton (A27L) Fusion Protein Forms a Triple Coiled-Coil Structure and Interacts with the 21-Kilodalton (A17L) Virus Membrane Protein through a C-Terminal α-Helix

1998 ◽  
Vol 72 (12) ◽  
pp. 10126-10137 ◽  
Author(s):  
María-Isabel Vázquez ◽  
German Rivas ◽  
David Cregut ◽  
Luis Serrano ◽  
Mariano Esteban
Keyword(s):  
Amino Acids ◽  
Vaccinia Virus ◽  
Structural Model ◽  
Analytical Ultracentrifugation ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
C Terminus ◽  
Α Helix ◽  
Mutant Forms ◽  
Helical Region

ABSTRACT The vaccinia virus 14-kDa protein (encoded by the A27L gene) plays an important role in the biology of the virus, acting in virus-to-cell and cell-to-cell fusions. The protein is located on the surface of the intracellular mature virus form and is essential for both the release of extracellular enveloped virus from the cells and virus spread. Sequence analysis predicts the existence of four regions in this protein: a structureless region from amino acids 1 to 28, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Circular dichroism spectroscopy, analytical ultracentrifugation, and chemical cross-linking studies of the purified wild-type protein and several mutant forms, lacking one or more of the above regions or with point mutations, support the above-described structural division of the 14-kDa protein. The two contiguous cysteine residues at positions 71 and 72 are not responsible for the formation of 14-kDa protein trimers. The location of hydrophobic residues at the a and d positions on a helical wheel and of charged amino acids in adjacent positions, e and g, suggests that the hydrophobic and ionic interactions in the triple coiled-coil helical region are involved in oligomer formation. This conjecture was supported by the construction of a three-helix bundle model and molecular dynamics. Binding assays with purified proteins expressed in Escherichia coli and cytoplasmic extracts from cells infected with a virus that does not produce the 14-kDa protein during infection (VVindA27L) show that the 21-kDa protein (encoded by the A17L gene) is the specific viral binding partner and identify the putative Leu zipper, the predicted third α-helix on the C terminus of the 14-kDa protein, as the region involved in protein binding. These findings were confirmed in vivo, following transfection of animal cells with plasmid vectors expressing mutant forms of the 14-kDa protein and infected with VVindA27L. We find the structural organization of 14kDa to be similar to that of other fusion proteins, such as hemagglutinin of influenza virus and gp41 of human immunodeficiency virus, except for the presence of a protein-anchoring domain instead of a transmembrane domain. Based on our observations, we have established a structural model of the 14-kDa protein.

Differential Mapping of the Amino Acids Mediating Agonist and Antagonist Coordination with the Human Thromboxane A2 Receptor Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3571-3571
Author(s):  
Fadi T. Khasawneh ◽  
Jin-Sheng Huang ◽  
Joseph W. Turek ◽  
Guy C. Le Breton
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
Functional Response ◽  
Transmembrane Domain ◽  
Thromboxane A2 ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Ligand Binding Pocket ◽  
Thromboxane A2 Receptor ◽  
Ligand Coordination

Abstract Despite the well-documented involvement of thromboxane A2 receptor (TPR) signaling in the pathogenesis of thrombotic diseases, there are currently no rationally-designed antagonists available for clinical use. To a large extent this derives from a lack of knowledge regarding the topography of the TPR ligand binding pocket. On this basis, the purpose of the current study was to identify the specific amino acid residues in the TPR protein which regulate ligand coordination and binding. The sites selected for mutation reside within or in close proximity to a region we previously defined as a TPR ligand binding site, i.e., the C-terminus of the second extracellular loop and the leading edge of the fifth transmembrane domain. Mutation of these residues caused varying effects on the TPR-ligand coordination process. Specifically, the D193A mutant lacked both SQ29,548 (antagonist) binding and U46619 (agonist)-induced calcium mobilization. Three other mutants, F184Y, T186A and S191T, discriminated between SQ29,548 binding and the U46619-mediated functional response. Furthermore, these mutants also revealed a divergence in the binding of two structurally different antagonists, SQ29,548 and BM13.505. Conversely, two separate mutants which exhibited SQ29,548 binding activity yielded either a normal (F196Y) or reduced (S201T) U46619 response. Finally, mutation of other residues directly adjacent to those described above, e.g., E190A and F200A, produced no detectable effects on either SQ29,548 binding or the U46619-induced functional response. In summary, these results identify key amino acids involved in TPR ligand coordination and demonstrate that TPR-specific ligands do not necessarily interact with the same residues in the ligand-binding pocket.

scholarly journals LsbB Bacteriocin Interacts with the Third Transmembrane Domain of the YvjB Receptor

2016 ◽  
Vol 82 (17) ◽  
pp. 5364-5374 ◽  
Author(s):  
Marija Miljkovic ◽  
Gordana Uzelac ◽  
Nemanja Mirkovic ◽  
Giulia Devescovi ◽  
Dzung B. Diep ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Sugar Transport ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Bacteriocin Activity ◽  
Binding Studies ◽  
Content Type ◽  
The Third

ABSTRACTThe Zn-dependent membrane-located protease YvjB has previously been shown to serve as a target receptor for LsbB, a class II leaderless lactococcal bacteriocin. AlthoughyvjBis highly conserved in the genusLactococcus, the bacteriocin appears to be active only against the subspeciesL. lactissubsp.lactis. Comparative analysis of the YvjB proteins of a sensitive strain (YvjBMN) and a resistant strain (YvjBMG) showed that they differ from each other in 31 positions. In this study, we applied site-directed mutagenesis and performed directed binding studies to provide biochemical evidence that LsbB interacts with the third transmembrane helix of YvjB in susceptible cells. The site-directed mutagenesis of LsbB and YvjB proteins showed that certain amino acids and the length of LsbB are responsible for the bacteriocin activity, most probably through adequate interaction of these two proteins; the essential amino acids in LsbB responsible for the activity are tryptophan (Trp25) and terminal alanine (Ala30). It was also shown that the distance between Trp25and terminal alanine is crucial for LsbB activity. The crucial region in YvjB for the interaction with LsbB is the beginning of the third transmembrane helix, particularly amino acids tyrosine (Tyr356) and alanine (Ala353).In vitroexperiments showed that LsbB could interact with both YvjBMNand YvjBMG, but the strength of interaction is significantly less with YvjBMG.In vivoexperiments with immunofluorescently labeled antibody demonstrated that LsbB specifically interacts only with cells carrying YvjBMN.IMPORTANCEThe antimicrobial activity of LsbB bacteriocin depends on the correct interaction with the corresponding receptor in the bacterial membrane of sensitive cells. Membrane-located bacteriocin receptors have essential primary functions, such as cell wall synthesis or sugar transport, and it seems that interaction with bacteriocins is suicidal for cells. This study showed that the C-terminal part of LsbB is crucial for the bacteriocin activity, most probably through adequate interaction with the third transmembrane domain of the YvjB receptor. The conserved Tyr356and Ala353residues of YvjB are essential for the function of this Zn-dependent membrane-located protease as a bacteriocin receptor.

scholarly journals Molecular Determinants of the Cofactor Specificity of Ribitol Dehydrogenase, a Short-Chain Dehydrogenase/Reductase

2012 ◽  
Vol 78 (9) ◽  
pp. 3079-3086 ◽  
Author(s):  
Hee-Jung Moon ◽  
Manish Kumar Tiwari ◽  
Ranjitha Singh ◽  
Yun Chan Kang ◽  
Jung-Kul Lee
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Content Type ◽  
Cofactor Specificity ◽  
Charged Amino Acids ◽  
Molecular Determinants ◽  
Ribitol Dehydrogenase

ABSTRACTRibitol dehydrogenase fromZymomonas mobilis(ZmRDH) catalyzes the conversion of ribitol tod-ribulose and concomitantly reduces NAD(P)+to NAD(P)H. A systematic approach involving an initial sequence alignment-based residue screening, followed by a homology model-based screening and site-directed mutagenesis of the screened residues, was used to study the molecular determinants of the cofactor specificity of ZmRDH. A homologous conserved amino acid, Ser156, in the substrate-binding pocket of the wild-type ZmRDH was identified as an important residue affecting the cofactor specificity of ZmRDH. Further insights into the function of the Ser156 residue were obtained by substituting it with other hydrophobic nonpolar or polar amino acids. Substituting Ser156 with the negatively charged amino acids (Asp and Glu) altered the cofactor specificity of ZmRDH toward NAD+(S156D, [kcat/Km,NAD]/[kcat/Km,NADP] = 10.9, whereKm,NADis theKmfor NAD+andKm,NADPis theKmfor NADP+). In contrast, the mutants containing positively charged amino acids (His, Lys, or Arg) at position 156 showed a higher efficiency with NADP+as the cofactor (S156H, [kcat/Km,NAD]/[kcat/Km,NADP] = 0.11). These data, in addition to those of molecular dynamics and isothermal titration calorimetry studies, suggest that the cofactor specificity of ZmRDH can be modulated by manipulating the amino acid residue at position 156.

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