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1
Bell, J. K., I. Botos, P. R. Hall, J. Askins, J. Shiloach, D. M. Segal, and D. R. Davies. "The molecular structure of the Toll-like receptor 3 ligand-binding domain." Proceedings of the National Academy of Sciences 102, no. 31 (July 25, 2005): 10976–80. http://dx.doi.org/10.1073/pnas.0505077102.
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Shih, DT, JM Edelman, AF Horwitz, GB Grunwald, and CA Buck. "Structure/function analysis of the integrin beta 1 subunit by epitope mapping." Journal of Cell Biology 122, no. 6 (September 15, 1993): 1361–71. http://dx.doi.org/10.1083/jcb.122.6.1361.
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Monoclonal antibodies (mAbs) have been produced against the chicken beta 1 subunit that affect integrin functions, including ligand binding, alpha subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric beta 1 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel anti-chicken beta 1 mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken beta 1 subunit by homologous regions of the mouse beta 1 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse beta 1 domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken beta 1 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the beta 1 subunit. This region is homologous to a portion of the ligand-binding domain of the beta 3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce alpha/beta subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of beta 1 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the alpha subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the beta 1 subunit.
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Jensen, Maria Risager, Goran Bajic, Xianwei Zhang, Anne Kjær Laustsen, Heidi Koldsø, Katrine Kirkeby Skeby, Birgit Schiøtt, Gregers R. Andersen, and Thomas Vorup-Jensen. "Structural Basis for Simvastatin Competitive Antagonism of Complement Receptor 3." Journal of Biological Chemistry 291, no. 33 (June 23, 2016): 16963–76. http://dx.doi.org/10.1074/jbc.m116.732222.
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The complement system is an important part of the innate immune response to infection but may also cause severe complications during inflammation. Small molecule antagonists to complement receptor 3 (CR3) have been widely sought, but a structural basis for their mode of action is not available. We report here on the structure of the human CR3 ligand-binding I domain in complex with simvastatin. Simvastatin targets the metal ion-dependent adhesion site of the open, ligand-binding conformation of the CR3 I domain by direct contact with the chelated Mg2+ ion. Simvastatin antagonizes I domain binding to the complement fragments iC3b and C3d but not to intercellular adhesion molecule-1. By virtue of the I domain's wide distribution in binding kinetics to ligands, it was possible to identify ligand binding kinetics as discriminator for simvastatin antagonism. In static cellular experiments, 15–25 μm simvastatin reduced adhesion by K562 cells expressing recombinant CR3 and by primary human monocytes, with an endogenous expression of this receptor. Application of force to adhering monocytes potentiated the effects of simvastatin where only a 50–100 nm concentration of the drug reduced the adhesion by 20–40% compared with untreated cells. The ability of simvastatin to target CR3 in its ligand binding-activated conformation is a novel mechanism to explain the known anti-inflammatory effects of this compound, in particular because this CR3 conformation is found in pro-inflammatory environments. Our report points to new designs of CR3 antagonists and opens new perspectives and identifies druggable receptors from characterization of the ligand binding kinetics in the presence of antagonists.
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Springer, Timothy A., Junichi Takagi, Barry S. Coller, Jia-Huai Wang, and Tsan Xiao. "Crystal Structure of the Integrin αIIBβ3 Headpiece at 2.7–3.1 Å: Structure, Mechanisms of Activation and Ligand Binding, Inhibition by Eptifibatide, Tirofiban, and mAb 10E5, and Structure of the HPA-1 Alloantigen Epitope." Blood 104, no. 11 (November 16, 2004): 327. http://dx.doi.org/10.1182/blood.v104.11.327.327.
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Abstract The αIIbβ3 headpiece (αIIb, 1–621; β3, 1–472) was expressed in CHO cells, purified, digested with chymotrypsin, mixed with either mAb 10E5 Fab (form A) or without (form B), repurified, digested with carboxypeptidase (leaving αIIb, 1–452 and β3, 1–440) and crystallized with PEG, Mg acetate, and Na cacodylate at 4°C. Cocrystallization of αIIbβ3/10E5 (A) with eptifibatide or tirofiban was with imidazole instead of cacodylate. Crystals were diffracted at APS and CHESS and analyzed by HKL2000, AMoRe, O, CNS, and CCP4 software. Crystal forms A and B contain 1 and 3 molecules/asymmetric unit (2.7–3.1 and 2.9 Å resolution), respectively. Ca2+ was assigned at the 4 αIIb β-hairpin sites in blades 4–7, and I-like (βA) LIMBS and ADMIDAS; Mg2+ was assigned to MIDAS. The major findings are: 1) As compared to unliganded αVβ3, αIIbβ3 has a ~62° outward pivot of the β3 hybrid domain from the I-like (βA) domain, indicating adoption of an open, high affinity conformation driven by cacodylate or the Asp (D) carboxyl of the drugs binding to MIDAS and acting as activating ligand equivalents. 2) The αIIb ligand binding cleft is rigid and includes αIIb D224 [end-on H bond to ligand Lys (K) or Arg (R)] and hydrophobic residues F160, Y190, and F231, accounting for the selective binding to αIIbβ3 (vs αVβ3) of KGD and homoarginine-GDW peptides, fibrinogen γ-chain peptide, eptifibatide, and tirofiban. 3) 10E5 Fab interacts with a unique “cap” subdomain in αIIb formed by 4 insertions in β-propeller loops in blades 1–3 that form a β-sheet and α-helix structure involved in ligand binding. 4) Comparison of unliganded αVβ3 and liganded αIIbβ3 indicates that receptor activation and ligand binding involves: extensive movement of β3 subunit β1-α1 loop and α1 helix, and β6-α7 loop and α7 helix; alterations in the coordinating residues at the ADMIDAS, MIDAS, and LIMBS; and breaking the ADMIDAS Ca2+ coordination by the M335 backbone carbonyl (providing a mechanism by which Mn2+, which competes with Ca2+ at the ADMIDAS but has a lower propensity for carbonyl coordination than Ca2+, activates integrins). The 62° pivot results from a one-turn piston-like displacement of the α7 helix involving a hydrophobic ratchet of the β6-α7 loop; a ratchet motion of the α1-helix in which L134 moves to the space previously occupied by V340; and complete remodeling at the interface between the β3 I-like (βA) and hybrid domains. 5) The structure of the β3 PSI domain reveals that the long range disulfide is between β3 C13 (rather than C5) and C435, and comparison to the PSI of semaphorin 4D demonstrates that C435 is an integral part of the PSI domain fold. Thus, the I-like (βA) domain appears to be inserted in the hybrid domain, which is inserted in the PSI domain. 6) The structure reveals the location of the Leu/Pro-33 PSI polymorphism responsible for the HPA1 alloantigen. At a rigid interface with the hybrid domain, polymorphism of Arg93 demonstrates the requirement of the hybrid/PSI interface for alloantigenicity at Leu-33. Overall, the structure reveals how allostery regulates ligand binding affinity of αIIbβ3, and how the outward swing of the lever-like hybrid and PSI domains communicates the conformation of the ligand binding site to the α and β leg domains, and to the membrane and cytosol.
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Kolenko, Petr, Daniel Rozbeský, Tereza Skálová, Tomáš Kovaľ, Karla Fejfarová, Jarmila Dušková, Jan Stránský, Jindřich Hašek, and Jan Dohnálek. "Domain swapping in structure of mNKR-P1A: unique feature with unknown function." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C249. http://dx.doi.org/10.1107/s2053273314097502.
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Natural killer (NK) cells, large granular lymphocytes, play an important role in the innate immune response against viruses, parasites and tumour cells. NK cells use a wide repertoire of surface receptors to modulate their activity [1]. The family of NKR-P1 surface receptors of NK cells belong to proteins with C-type lectin-like (CTL) fold. The overall architecture of other known CTL receptors (e.g. members of Ly49 family, NKG2D, CD94, mouse CLRg) is conserved [2]. The mechanism of ligand binding has been revealed by the crystal structure of Nkp65 bound to its keratinocyte ligand [3]. However, observation of domain swapping in crystal structure of mouse (m) NKR-P1A represents an unusual structural feature that might be involved in a new mechanism of ligand binding that would be specific for some members of NKR-P1 family. Nevertheless, our crystal structure of mNKR-P1A represents a unique structural observation that demands careful analysis. Even the latest structural studies do not answer the question of function or role of swapped domain of the receptor in potential ligand binding. We have generated new variants of mNKR-P1A of varied chain length that undergo biochemical and structural analysis including mass spectrometry.
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Chen, Yang, Joakim Näsvall, Shiying Wu, Dan I. Andersson, and Maria Selmer. "Structure of AadA fromSalmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase." Acta Crystallographica Section D Biological Crystallography 71, no. 11 (October 31, 2015): 2267–77. http://dx.doi.org/10.1107/s1399004715016429.
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Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA fromSalmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis.In vivoresistance andin vitrobinding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.
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Li, Chaoqun, Xiaojia Zhao, Xiaomin Zhu, Pengtao Xie, and Guangju Chen. "Structural Studies of the 3′,3′-cGAMP Riboswitch Induced by Cognate and Noncognate Ligands Using Molecular Dynamics Simulation." International Journal of Molecular Sciences 19, no. 11 (November 9, 2018): 3527. http://dx.doi.org/10.3390/ijms19113527.
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Riboswtich RNAs can control gene expression through the structural change induced by the corresponding small-molecule ligands. Molecular dynamics simulations and free energy calculations on the aptamer domain of the 3′,3′-cGAMP riboswitch in the ligand-free, cognate-bound and noncognate-bound states were performed to investigate the structural features of the 3′,3′-cGAMP riboswitch induced by the 3′,3′-cGAMP ligand and the specificity of ligand recognition. The results revealed that the aptamer of the 3′,3′-cGAMP riboswitch in the ligand-free state has a smaller binding pocket and a relatively compact structure versus that in the 3′,3′-cGAMP-bound state. The binding of the 3′,3′-cGAMP molecule to the 3′,3′-cGAMP riboswitch induces the rotation of P1 helix through the allosteric communication from the binding sites pocket containing the J1/2, J1/3 and J2/3 junction to the P1 helix. Simultaneously, these simulations also revealed that the preferential binding of the 3′,3′-cGAMP riboswitch to its cognate ligand, 3′,3′-cGAMP, over its noncognate ligand, c-di-GMP and c-di-AMP. The J1/2 junction in the 3′,3′-cGAMP riboswitch contributing to the specificity of ligand recognition have also been found.
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Rudenko, Gabby, Thai Nguyen, Yogarany Chelliah, Thomas C. Südhof, and Johann Deisenhofer. "Regulation of LNS Domain Function by Alternative Splicing: The Structure of the Ligand-Binding Domain of Neurexin Iβ." Cell 99, no. 1 (October 1999): 93–101. http://dx.doi.org/10.1016/s0092-8674(00)80065-3.
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Tamura, Tatsushiro, Jun Yamanouchi, Shigeru Fujita, and Takaaki Hato. "Critical residues for ligand binding in blade 2 of the propeller domain of the integrin αIIb subunit." Thrombosis and Haemostasis 91, no. 01 (2004): 111–18. http://dx.doi.org/10.1160/th03-06-0392.
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SummaryLigand binding to integrin αIIbβ3 is a key event of thrombus formation. The propeller domain of the αIIb subunit has been implicated in ligand binding. Recently, the ligand binding site of the αV propeller was determined by crystal structure analysis. However, the structural basis of ligand recognition by the αIIb propeller remains to be determined. In this study, we conducted site-directed mutagenesis of all residues located in the loops extending above blades 2 and 4 of the αIIb propeller, which are spatially close to, but distinct from, the loops that contain the binding site for an RGD ligand in the crystal structure of the αV propeller. Replacement by alanine of Q111, H112 or N114 in the loop within the blade 2 (the W2:2-3 loop in the propeller model) abolished binding of a ligand-mimetic antibody and fibrinogen to αIIbβ3 induced by different types of integrin activation including activation of αIIbβ3 by β3 cytoplasmic mutation. CHO cells stably expressing recombinant αIIbβ3 bearing Q111A, H112A or N114A mutation did not exhibit αIIbβ3mediated adhesion to fibrinogen. According to the crystal structure of αVβ3, the αV residue corresponding to αIIbN114 is exposed on the integrin surface and close to the RGD binding site. These results suggest that the Q111, H112 and N114 residues in the loop within blade 2 of the αIIb propeller are critical for ligand binding, possibly because of direct interaction with ligands or modulation of the RGD binding pocket.
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Rossjohn, Jamie, William J. McKinstry, Joanna M. Woodcock, Barbara J. McClure, Timothy R. Hercus, Michael W. Parker, Angel F. Lopez, and Christopher J. Bagley. "Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common β-chain bound to an antagonist." Blood 95, no. 8 (April 15, 2000): 2491–98. http://dx.doi.org/10.1182/blood.v95.8.2491.
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Abstract Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor β-chain (βc) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine421 (Tyr421), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr421, which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of βc also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems.
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Rossjohn, Jamie, William J. McKinstry, Joanna M. Woodcock, Barbara J. McClure, Timothy R. Hercus, Michael W. Parker, Angel F. Lopez, and Christopher J. Bagley. "Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common β-chain bound to an antagonist." Blood 95, no. 8 (April 15, 2000): 2491–98. http://dx.doi.org/10.1182/blood.v95.8.2491.008k06_2491_2498.
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Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor β-chain (βc) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine421 (Tyr421), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr421, which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of βc also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems.
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Sayre, P. H., R. E. Hussey, H. C. Chang, T. L. Ciardelli, and E. L. Reinherz. "Structural and binding analysis of a two domain extracellular CD2 molecule." Journal of Experimental Medicine 169, no. 3 (March 1, 1989): 995–1009. http://dx.doi.org/10.1084/jem.169.3.995.
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The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.
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Boesen, Christian C., Sergei Radaev, Shawn A. Motyka, Apisit Patamawenu, and Peter D. Sun. "The 1.1 Å Crystal Structure of Human TGF-β Type II Receptor Ligand Binding Domain." Structure 10, no. 7 (July 2002): 913–19. http://dx.doi.org/10.1016/s0969-2126(02)00780-3.
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Laursen, Louise, Elin Karlsson, Stefano Gianni, and Per Jemth. "Functional interplay between protein domains in a supramodular structure involving the postsynaptic density protein PSD-95." Journal of Biological Chemistry 295, no. 7 (December 12, 2019): 1992–2000. http://dx.doi.org/10.1074/jbc.ra119.011050.
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Cell scaffolding and signaling are governed by protein–protein interactions. Although a particular interaction is often defined by two specific domains binding to each other, this interaction often occurs in the context of other domains in multidomain proteins. How such adjacent domains form supertertiary structures and modulate protein–protein interactions has only recently been addressed and is incompletely understood. The postsynaptic density protein PSD-95 contains a three-domain supramodule, denoted PSG, which consists of PDZ, Src homology 3 (SH3), and guanylate kinase-like domains. The PDZ domain binds to the C terminus of its proposed natural ligand, CXXC repeat–containing interactor of PDZ3 domain (CRIPT), and results from previous experiments using only the isolated PDZ domain are consistent with the simplest scenario for a protein–protein interaction; namely, a two-state mechanism. Here we analyzed the binding kinetics of the PSG supramodule with CRIPT. We show that PSG binds CRIPT via a more complex mechanism involving two conformational states interconverting on the second timescale. Both conformational states bound a CRIPT peptide with similar affinities but with different rates, and the distribution of the two conformational states was slightly shifted upon CRIPT binding. Our results are consistent with recent structural findings of conformational changes in PSD-95 and demonstrate how conformational transitions in supertertiary structures can shape the ligand-binding energy landscape and modulate protein–protein interactions.
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Fernandes, Humberto, Honorata Czapinska, Katarzyna Grudziaz, Janusz M. Bujnicki, and Martyna Nowacka. "Crystal structure of human Acinus RNA recognition motif domain." PeerJ 6 (July 4, 2018): e5163. http://dx.doi.org/10.7717/peerj.5163.
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Acinus is an abundant nuclear protein involved in apoptosis and splicing. It has been implicated in inducing apoptotic chromatin condensation and DNA fragmentation during programmed cell death. Acinus undergoes activation by proteolytic cleavage that produces a truncated p17 form that comprises only the RNA recognition motif (RRM) domain. We have determined the crystal structure of the human Acinus RRM domain (AcRRM) at 1.65 Å resolution. It shows a classical four-stranded antiparallel β-sheet fold with two flanking α-helices and an additional, non-classical α-helix at the C-terminus, which harbors the caspase-3 target sequence that is cleaved during Acinus activation. In the structure, the C-terminal α-helix partially occludes the potential ligand binding surface of the β-sheet and hypothetically shields it from non-sequence specific interactions with RNA. Based on the comparison with other RRM-RNA complex structures, it is likely that the C-terminal α-helix changes its conformation with respect to the RRM core in order to enable RNA binding by Acinus.
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Smaldone, Giovanni, Alessia Ruggiero, Nicole Balasco, and Luigi Vitagliano. "Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima." International Journal of Molecular Sciences 21, no. 20 (October 12, 2020): 7503. http://dx.doi.org/10.3390/ijms21207503.
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Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.
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Akke, Mikael. "Conformational dynamics and thermodynamics of protein–ligand binding studied by NMR relaxation." Biochemical Society Transactions 40, no. 2 (March 21, 2012): 419–23. http://dx.doi.org/10.1042/bst20110750.
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Protein conformational dynamics can be critical for ligand binding in two ways that relate to kinetics and thermodynamics respectively. First, conformational transitions between different substates can control access to the binding site (kinetics). Secondly, differences between free and ligand-bound states in their conformational fluctuations contribute to the entropy of ligand binding (thermodynamics). In the present paper, I focus on the second topic, summarizing our recent results on the role of conformational entropy in ligand binding to Gal3C (the carbohydrate-recognition domain of galectin-3). NMR relaxation experiments provide a unique probe of conformational entropy by characterizing bond-vector fluctuations at atomic resolution. By monitoring differences between the free and ligand-bound states in their backbone and side chain order parameters, we have estimated the contributions from conformational entropy to the free energy of binding. Overall, the conformational entropy of Gal3C increases upon ligand binding, thereby contributing favourably to the binding affinity. Comparisons with the results from isothermal titration calorimetry indicate that the conformational entropy is comparable in magnitude to the enthalpy of binding. Furthermore, there are significant differences in the dynamic response to binding of different ligands, despite the fact that the protein structure is virtually identical in the different protein–ligand complexes. Thus both affinity and specificity of ligand binding to Gal3C appear to depend in part on subtle differences in the conformational fluctuations that reflect the complex interplay between structure, dynamics and ligand interactions.
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Wright, P. S., V. Saudek, T. J. Owen, S. L. Harbeson, and A. J. Bitonti. "An echistatin C-terminal peptide activates GPIIbIIIa binding to fibrinogen, fibronectin, vitronectin and collagen type I and type IV." Biochemical Journal 293, no. 1 (July 1, 1993): 263–67. http://dx.doi.org/10.1042/bj2930263.
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Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.
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Akçeşme, Faruk Berat, Nail Beşli, Jorge Peña-García, and Horacio Pérez-Sánchez. "Assessment of Interaction of Human OCT 1-3 Proteins and Metformin Using Silico Analyses." Acta Chimica Slovenica 67, no. 4 (December 15, 2020): 1202–15. http://dx.doi.org/10.17344/acsi.2020.6108.
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Metformin, a drug frequently used by diabetic patients as the first-line treatment worldwide, is positively charged and is transported into the cell through human organic cation transporter (hOCT 1-3) proteins. We aimed to mimic the cellular uptake of metformin by hOCT1-3 with various bioinformatics methods and tools. 3D structure of OCT1-3 proteins was predicted by considering the structures and function of these proteins. We predicted functional regions (active and ligand binding sites) of OCT1-3 and performed comparative bioinformatics analysis. The predicted structure of hOCT1-3 was then analyzed in the Blind Docking server and the results were confirmed with predicted binding site residues and conserved domain regions. We simulated the OCT1-3 and metformin docking and also validated the docking procedure with other substrates of HOCT1-3 proteins. We selected the best poses of metformin docking simulations as per binding energy (–5.27 to –4.60 kcal/mol). Lastly, we validated the static description of protein-ligand (OCT-Metformin) interactions by performing molecular dynamics simulation. Eventually, we obtained stable simulation of OCT-metformin interaction.
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Byrne, Lee J., Ateesh Sidhu, A. Katrine Wallis, Lloyd W. Ruddock, Robert B. Freedman, Mark J. Howard, and Richard A. Williamson. "Mapping of the ligand-binding site on the b′ domain of human PDI: interaction with peptide ligands and the x-linker region." Biochemical Journal 423, no. 2 (September 25, 2009): 209–17. http://dx.doi.org/10.1042/bj20090565.
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PDI (protein disulfide-isomerase) catalyses the formation of native disulfide bonds of secretory proteins in the endoplasmic reticulum. PDI consists of four thioredoxin-like domains, of which two contain redox-active catalytic sites (a and a′), and two do not (b and b′). The b′ domain is primarily responsible for substrate binding, although the nature and specificity of the substrate-binding site is still poorly understood. In the present study, we show that the b′ domain of human PDI is in conformational exchange, but that its structure is stabilized by the addition of peptide ligands or by binding the x-linker region. The location of the ligand-binding site in b′ was mapped by NMR chemical shift perturbation and found to consist primarily of residues from the core β-sheet and α-helices 1 and 3. This site is where the x-linker region binds in the X-ray structure of b′x and we show that peptide ligands can compete with x binding at this site. The finding that x binds in the principal ligand-binding site of b′ further supports the hypothesis that x functions to gate access to this site and so modulates PDI activity.
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Booker, Grant W., Ivan Gout, A. Kristina^Downing, Paul C. Driscoll, Jonathan Boyd, Michael D. Waterfield, and Iain D. Campbell. "Solution structure and ligand-binding site of the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase." Cell 73, no. 4 (May 1993): 813–22. http://dx.doi.org/10.1016/0092-8674(93)90259-s.
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Fujiwara, Yoshie, Natsuko Goda, Tomonari Tamashiro, Hirotaka Narita, Kaori Satomura, Takeshi Tenno, Atsushi Nakagawa, et al. "Crystal structure of afadin PDZ domain-nectin-3 complex shows the structural plasticity of the ligand-binding site." Protein Science 24, no. 3 (January 13, 2015): 376–85. http://dx.doi.org/10.1002/pro.2628.
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Yu, Xinzhe, and Ping Yi. "Structural Insights of Transcriptionally Active, Full-Length Androgen Receptor Coactivator Complexes." Journal of the Endocrine Society 5, Supplement_1 (May 1, 2021): A817. http://dx.doi.org/10.1210/jendso/bvab048.1665.
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Abstract Steroid hormone receptors activate gene transcription by binding specific DNA sequences and recruiting coactivators to initiate transcription of their target genes. For most nuclear hormone receptors (NRs), the ligand-dependent activation function domain-2 (AF-2), residing in the C-terminal ligand binding domain (LBD), is a primary contributor to the NR transcriptional activity. In contrast to other steroid receptors such as estrogen receptor-α (ERα), the transcriptional activation function of androgen receptor (AR) is thought to be largely dependent on its ligand-independent activation function domain-1 (AF-1) located in its N-terminal domain (NTD). It remains unclear why AR utilizes a different activation function domain from other steroid receptors despite the fact that NRs share similar domain organizations and have a highly conserved DNA binding domain (DBD) and LBD. Here we present cryo-electron microscopic structures of DNA-bound full-length AR and its functional complex structure with its key coactivators, steroid receptor coactivator-3 (SRC-3) and the histone acetyltransferase p300. The structures reveal that androgen-bound full-length AR dimerization follows a unique head-to-head and tail-to-tail manner with all of the domains involved. The DBD and LBD are located at the center of the dimer interface. The NTDs wrap around the LBDs through their unique intra- and inter-molecular N- and C-terminal interactions and connect to each other. We observe that unlike ERα, AR binds a single SRC-3 molecule along with a single p300 molecule. The AR NTD appears to be the primary site for recruitment of both coactivators. The N-terminal region of SRC-3, rather than its receptor interaction domain, is important for this interaction. The structures presented here highlight the importance of the AR NTD for its transcriptional functions and provide a structural basis for understanding the assembly of the AR:coactivator complex and its domain contributions to transcriptional regulation.
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Sharma, Tripti. "INSILICO DOCKING APPROACH TO STUDY THE BINDING AFFINITY OF ISOFLAVONES ON THE CRYSTAL STRUCTURE OF ESTROGEN RECEPTOR ALPHA." INDIAN DRUGS 54, no. 10 (October 28, 2017): 7–15. http://dx.doi.org/10.53879/id.54.10.11152.
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The objective of the study was to carry out docking studies of isoflavone derivatives and examine their binding efficiencies to the ligand binding domain of ERα using Autodock program. A series of isoflavone derivatives were computationally designed and optimized with the AutoDock Vina software to investigate the interactions between the target compounds and the amino acid residues of the ERα.. In silico docking studies were carried out using AutoDock Vina, based on the Lamarckian genetics algorithm principle. The results showed that all the selected isoflavones showed binding energy ranging between -7.44 kcal/mol to -10.1 kcal/mol, when compared with that of the standard compound tamoxifen (-10.0 kcal/mol). Among all the designed compounds, 3-[3-(naphthalen-2-yl) phenyl]-2, 3-dihydro-4Hchroman- 4-one (Compound 12) shows more binding energy values (-10.1 kcal/mol). The present findings provide valuable information on the binding process of Isoflavones compounds to the binding site of ERα and reveal the structural requirement needed for binding.
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Yong, E. "Partial androgen insensitivity and correlations with the predicted three dimensional structure of the androgen receptor ligand-binding domain." Molecular and Cellular Endocrinology 137, no. 1 (February 13, 1998): 41–50. http://dx.doi.org/10.1016/s0303-7207(97)00229-3.
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Suino-Powell, Kelly, Yong Xu, Chenghai Zhang, Yong-guang Tao, W. David Tolbert, S. Stoney Simons, and H. Eric Xu. "Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol." Molecular and Cellular Biology 28, no. 6 (December 26, 2007): 1915–23. http://dx.doi.org/10.1128/mcb.01541-07.
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ABSTRACT A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GR LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 Å3, effectively doubling the size of the GR dexamethasone-binding pocket of 540 Å3 and yet leaving the structure of the coactivator binding site intact. DAC occupies only ∼50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.
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Sakurai, Shunya, Umeharu Ohto, and Toshiyuki Shimizu. "Structure of human Roquin-2 and its complex with constitutive-decay element RNA." Acta Crystallographica Section F Structural Biology Communications 71, no. 8 (July 29, 2015): 1048–54. http://dx.doi.org/10.1107/s2053230x15011887.
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Roquin mediates mRNA degradation by recognizing the constitutive-decay element (CDE) in the 3′ untranslated region of the target gene followed by recruitment of the deadenylation machinery. Deficiency or dysfunction of Roquin has been associated with autoimmunity and inflammation. To establish the structural basis for the recognition of CDE RNA by Roquin, the crystal structure of the ROQ domain of human Roquin-2 was determined in ligand-free and CDE-derived RNA-bound forms. The ROQ domain of Roquin-2 folded into a winged-helix structure in which the wing region showed structural flexibility and acted as a lid for RNA binding. The CDE RNA, forming a stem-loop structure, bound to the positively charged surface of the ROQ domain and was mainly recognizedviadirect interactions with the phosphate backbone in the 5′ half of the stem-loop and its triloop andviaindirect water-mediated interactions. Structural comparison with Roquin-1 revealed conserved features of the RNA-binding mode. Therefore, it is suggested that the Roquin proteins function redundantly in mRNA degradation.
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Oliveira, Laerte, Claudio M. Costa-Neto, Clovis R. Nakaie, Shirley Schreier, Suma I. Shimuta, and Antonio C. M. Paiva. "The Angiotensin II AT1 Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure." Physiological Reviews 87, no. 2 (April 2007): 565–92. http://dx.doi.org/10.1152/physrev.00040.2005.
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The most prevalent physiological effects of ANG II, the main product of the renin-angiotensin system, are mediated by the AT1 receptor, a rhodopsin-like AGPCR. Numerous studies of the cardiovascular effects of synthetic peptide analogs allowed a detailed mapping of ANG II's structural requirements for receptor binding and activation, which were complemented by site-directed mutagenesis studies on the AT1 receptor to investigate the role of its structure in ligand binding, signal transduction, phosphorylation, binding to arrestins, internalization, desensitization, tachyphylaxis, and other properties. The knowledge of the high-resolution structure of rhodopsin allowed homology modeling of the AT1 receptor. The models thus built and mutagenesis data indicate that physiological (agonist binding) or constitutive (mutated receptor) activation may involve different degrees of expansion of the receptor's central cavity. Residues in ANG II structure seem to control these conformational changes and to dictate the type of cytosolic event elicited during the activation. 1) Agonist aromatic residues (Phe8 and Tyr4) favor the coupling to G protein, and 2) absence of these residues can favor a mechanism leading directly to receptor internalization via phosphorylation by specific kinases of the receptor's COOH-terminal Ser and Thr residues, arrestin binding, and clathrin-dependent coated-pit vesicles. On the other hand, the NH2-terminal residues of the agonists ANG II and [Sar1]-ANG II were found to bind by two distinct modes to the AT1 receptor extracellular site flanked by the COOH-terminal segments of the EC-3 loop and the NH2-terminal domain. Since the [Sar1]-ligand is the most potent molecule to trigger tachyphylaxis in AT1 receptors, it was suggested that its corresponding binding mode might be associated with this special condition of receptors.
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Slater, Alexandre, Ying Di, Joanne C. Clark, Natalie J. Jooss, Eleyna M. Martin, Fawaz Alenazy, Mark R. Thomas, et al. "Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer." Blood 137, no. 24 (June 17, 2021): 3443–53. http://dx.doi.org/10.1182/blood.2020009440.
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Abstract Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C′ loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C′ loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
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Ramos, Jorge, Wonyong Jung, Josefina Ramos-Franco, Gregory A. Mignery, and Michael Fill. "Single Channel Function of Inositol 1,4,5-trisphosphate Receptor Type-1 and -2 Isoform Domain-Swap Chimeras." Journal of General Physiology 121, no. 5 (April 14, 2003): 399–411. http://dx.doi.org/10.1085/jgp.200208718.
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The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.
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Boggon, Titus J. "Jak3 Kinase Domain Crystal Structures and Implications for Jak-Specific Drug Design." Blood 106, no. 11 (November 16, 2005): 69. http://dx.doi.org/10.1182/blood.v106.11.69.69.
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Abstract Janus (Jak) family non-receptor tyrosine kinases are critical for appropriate signaling of many growth factors and cytokines. The four vertebrate Jak kinase family members demonstrate differential receptor cytoplasmic tail binding associations and transduce discrete signals on extracellular binding of ligand to the transmembrane cytokine or growth factor receptor. On ligand binding, a rapid tyrosine phosphorylation mediated signaling cascade is initiated, culminating in translocation of cytosolic latent transcription factors, signal transducer and activator of transcription (Stat) proteins, to the nucleus and targeted activation of transcription. Dysregulation of this Jak-mediated signaling pathway is documented in a number of hematological diseases: Improper upregulation of Jak activity is seen in certain hematological malignancies [1] and inability to appropriately transduce signals from the common gamma chain, γc, through Jak3 is responsible for approximately 60% of human severe combined immunodeficiency cases [2]. In addition, Jak2 point mutation V617F is frequently documented in myeloproliferative disorders including polycythemia vera; this activating mutation may disrupt an autoinhibited conformation [3]. Therapies targeting restraint of Jak family tyrosine kinase activity may be useful for treating inappropriate activation of Jak signaling cascades or for suppressing the immune response. Advances towards structure-directed drug design of Jak-specific inhibitors were made recently with solution of the Jak3 kinase domain X-ray crystal structure [4], representing the first three-dimensional structural data for any portion of the Jak family of tyrosine kinases. Here three further crystal structures of the kinase domain of Jak3 are presented: an improved resolution co-crystal structure with staurosporine analog AFN-941 and two crystal forms of Jak3 kinase domain in complex with the kinase inhibitor small molecule compound QAD-409. Comparisons between these three solved Jak3 kinase domain crystal structures illustrate conformational flexibility between the kinase domain lobes and in the area of the catalytic cleft. Further structure analysis is also presented documenting in silico modeling of the binding of small molecule CP-690,550 [5] to the different Jak3 kinase domain crystal forms. Potential binding conformations of this inhibitor to the Jak3 kinase domain are suggested with one highly scored binding conformation predicted for all crystal forms. The crystal structures and modeling studies presented further define the extent of the Jak kinase catalytic cleft, demonstrate conformational plasticity in the active conformation Jak3 kinase domain and will aid the design of higher specificity Jak inhibitors.
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Kiran, K. S., M. K. Kokila, Guruprasad R, and Niranjan M.S. "Crystal Structure Determination and Molecular Docking Studies of 4- (5-Phenyl Pyrazin-2-Yl)-4h-1,2,4 Triazole-3-Thiol with Focal Adhesion Kinase Inhibitors." Open Chemistry Journal 3, no. 1 (September 15, 2016): 69–74. http://dx.doi.org/10.2174/1874842201603010069.
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The main objective of the present work is to determine crystal structure of the ligand by x-ray methods and to perform molecular docking studies of the ligand 4- Phenyl -5-Pyrazinyl-3-mercapto 1,2,4 Triazole with protein focal adhesion kinase (FAK) domain using the software, Autodock and pymol. Macromolecular modeling by docking studies provides the most detailed view possible of drug receptor interaction. It has created a new rational approach to drug design, where the structure of drug is designed, based on its fit to three dimensional structures of receptor site, rather than basing it on analogies to other active structures. The above titled compound is binding with FAK protein. This may act as inhibitor to FAK and can be used for anticancer therapy target.
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Radaeva, Mariia, Huifang Li, Eric LeBlanc, Kush Dalal, Fuqiang Ban, Fabrice Ciesielski, Bonny Chow, et al. "Structure-Based Study to Overcome Cross-Reactivity of Novel Androgen Receptor Inhibitors." Cells 11, no. 18 (September 7, 2022): 2785. http://dx.doi.org/10.3390/cells11182785.
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The mutation-driven transformation of clinical anti-androgen drugs into agonists of the human androgen receptor (AR) represents a major challenge for the treatment of prostate cancer patients. To address this challenge, we have developed a novel class of inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the androgen binding site (ABS) targeted by all conventional anti-AR drugs and prone to resistant mutations. While many members of the developed 4-(4-phenylthiazol-2-yl)morpholine series of AR-DBD inhibitors demonstrated the effective suppression of wild-type AR, a few represented by 4-(4-(3-fluoro-2-methoxyphenyl)thiazol-2-yl)morpholine (VPC14368) exhibited a partial agonistic effect toward the mutated T878A form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the observed cross-reactivity, we co-crystallized the T878A mutated form of the AR ligand binding domain (LBD) with a bound VPC14368 molecule. Computational modelling revealed that helix 12 of AR undergoes a characteristic shift upon VPC14368 binding causing the agonistic behaviour. Based on the obtained structural data we then designed derivatives of VPC14368 to successfully eliminate the cross-reactivity towards the AR ABS, while maintaining significant anti-AR DBD potency.
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Howard, Barbara-Ann, George Thom, Ian Jeffrey, Dave Colthurst, David Knowles, and Catherine Prescott. "Fragmentation of the ribosome to investigate RNA–ligand interactions." Biochemistry and Cell Biology 73, no. 11-12 (December 1, 1995): 1161–66. http://dx.doi.org/10.1139/o95-125.
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RNA molecules perform a variety of important and diverse functions and, therefore, an understanding of their structure and interaction with proteins and ligands is essential. Large RNA molecules (for example, the ribosomal RNAs) are complex and hence reports describing their fragmentation into functional subdomains has provided a means for their detailed analysis. We present here an in vivo approach to study RNA–ligand interactions. This is based on the concept that an RNA fragment could mimic a drug-binding site present on the intact molecule. Overexpression of the fragment would sequester the drug thereby permitting the continued functioning of the ribosome and, thus, ensuring cell viability. Accordingly, a fragment of 16S rRNA encompassing the spectinomycin-binding domain in helix 34 (nucleotides 1046–1065 and 1191–1211) was cloned and in vivo expression resulted in drug resistance. Furthermore, an RNA fragment lacking flanking sequences to helix 34 was also selected from among a pool of random rRNA fragments and shown to confer spectinomycin resistance. A similar in vitro approach is also described for the analysis of rRNA molecules that interact with the yeast elongation factor 3 (EF-3).Key words: rRNA, spectinomycin, EF-3.
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Yang, Zhao, Zhi-Li Huang, and Ya-Xiong Tao. "Functions of DPLIY motif and helix 8 of human melanocortin-3 receptor." Journal of Molecular Endocrinology 55, no. 2 (July 28, 2015): 107–17. http://dx.doi.org/10.1530/jme-15-0116.
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The melanocortin-3 receptor (MC3R) is a member of the family A G protein-coupled receptors (GPCRs). The MC3R remains the most enigmatic of the melanocortin receptors with regard to its physiological functions, especially its role in energy homeostasis. The N/DPxxY motif and the eighth helix (helix 8) in the carboxyl terminus of GPCRs have been identified to be important for receptor expression, ligand binding, signal transduction and internalization. To gain a better understanding of the structure-function relationship of MC3R, we performed a systematic study of all 20 residues in this domain using alanine-scanning mutagenesis. We showed that although all mutants were expressed normally on the cell surface, eleven residues were important for ligand binding and one was indispensable for downstream cAMP generation. F347A showed constitutive activity in cAMP signaling while all the other mutants had normal basal activities. We studied the signaling capacity of nine mutants in the ERK1/2 signaling pathway. All of these mutants showed normal basal ERK1/2 phosphorylation levels. The pERK1/2 levels of six binding- or signaling-defective mutants were enhanced upon agonist stimulation. The unbalanced cAMP and pERK1/2 signaling pathways suggested the existence of biased signaling in MC3R mutants. In summary, we showed that the DPLIY motif and helix 8 was important for MC3R activation and signal transduction. Our data led to a better understanding of the structure-function relationship of MC3R.
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COUETTE, Brigitte, Jérôme FAGART, Stéphan JALAGUIER, Marc LOMBES, Anny SOUQUE, and Marie-Edith RAFESTIN-OBLIN. "Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure." Biochemical Journal 315, no. 2 (April 15, 1996): 421–27. http://dx.doi.org/10.1042/bj3150421.
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To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) of hMR (hMR 711–984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone–hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1–944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711–984 region is sufficient for hsp90–MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand.
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Tweardy, David John, Xu Xuejun, Naijie Jing, and Huang Shao. "Structural Determinants for Signal Transducer and Activator of Transcription (STAT) 3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) at Phosphotyrosine Ligands 704 and 744." Blood 104, no. 11 (November 16, 2004): 2169. http://dx.doi.org/10.1182/blood.v104.11.2169.2169.
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Abstract Four tyrosine (Y) residues within the cytoplasmic domain of the G-CSFR (Y704, Y228, Y744 and Y764 in the human receptor; Y703, Y227, Y743 and Y763 in the murine receptor) become phosphorylated by Jak kinases upon ligand binding leading to recruitment of Src homology (SH) 2 domain-containing proteins that link to programs for myeloid cell survival and differentiation (Stat3 recruitment to Y704 and Y744) and proliferation (SHP-2 and PI3K recruitment to Y04; Grb2, Shc and SHP-2 recruitment to Y764). While the preference of SH2 domain binding to specific phospho (p) Y peptide ligands was shown to map to the three residues immediately C-terminal to the pY (+1, +2, +3 residues), the structural basis for these preferences is poorly understood but could be exploited to specifically target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation, which has been demonstrated in a subset of acute myelogenous leukemia (AML) patients whose cells contain Flt3 internal tandem duplications and who suffer relapse following initial chemotherapy. To establish the structural basis for Stat3 recruitment and activation by the G-CSFR at Y704 and Y744, we generated purified recombinant full-length Stat3 and phosphododecapeptides based on the sequence surrounding each Y within the G-CSFR. In peptide pull-down assays, recombinant Stat3 bound only to Y704 and Y744 phosphododecapeptide, which contain core pY motifs consisting of pYVLQ and pYLRC, respectively. In mirror resonance affinity assays employed to obtain quantitative binding information, Stat3 bound to each phosphododecapeptide with similar kinetics (e.g. KDs = 0.703 and 0.95 μM, respectively). We tested three models for Stat3 SH2-pY ligand binding proposed by us and others using wild type and mutant recombinant Stat3 proteins in peptide pull-down and mirror resonance affinity assays along with computer modeling of this interaction using the known structures of Statβ SH2 and EGFR pY ligand (EpY1068INQ). Our results revealed loss of binding of Stat3 to Y704 and Y744 phosphododecapeptides only in Stat3 mutated within the SH2 domain at K591 or R609, whose side chains interacted with the pY phosphate group, and in Stat3 mutated within the SH2 domain at E638, whose amide hydrogen bonded with oxygen within the +3 Q side chain (or with sulfur within the +3 C side chain) when the pY ligand assumes a β turn. G-CSF stimulation of cells co-expressing full-length G-CSFR and either wild type or mutant Stat3 constructs confirmed the requirements for the side chain of R609 and the amide hydrogen of E638 within the Stat3 SH2 domain for binding to the G-CSFR and subsequent phosphorylation of Stat3 on Y705. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of their interaction at Y704 and Y744 i.e. a β turn within the receptor pY motif and a key hydrogen bond formed between the polar side chain of the +3 residue and the amide hydrogen of E638 within the Stat3 SH2 domain. These features explain the preference of the Stat3 SH2 domain for pY peptide ligands with +3 Q or C as well as +3 T (pY705LKT within Stat3) and +3 S (pY743IRS within the murine G-CSFR) and can be exploited using a structure-assisted drug design strategy to develop new therapies for a subset of AML patients with poor prognosis whose cells demonstrate aberrant activation of Stat3.
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Cash, Devin R., Nicholas Noinaj, Susan K. Buchanan, and Cynthia Nau Cornelissen. "Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae." Infection and Immunity 83, no. 11 (September 8, 2015): 4438–49. http://dx.doi.org/10.1128/iai.00762-15.
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ABSTRACTNeisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects inEscherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue.
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Mankelow, Tosti J., Nicholas Burton, Fanney O. Stefansdottir, Frances A. Spring, Stephen F. Parsons, C. Fredrik Gilstring, R. Leo Brady, Mohandas Narla, Joel Anne Chasis, and David J. Anstee. "Characterisation of the Laminin 10/11 Binding Site on the Lutheran Glycoprotein Suggests a Novel Type of Protein-Protein Interaction." Blood 108, no. 11 (November 16, 2006): 1566. http://dx.doi.org/10.1182/blood.v108.11.1566.1566.
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Abstract The Lutheran glycoprotein is a five domain member of the immunoglobulin superfamily (IgSF) with a wide tissue distribution. It is a ligand for Laminin isoforms containing the alpha5 chain (Laminins 10 and 11). Lutheran glycoprotein on erythrocytes is thought to play a role in vasocclusive events that are a serious cause of morbidity in sickle cell anaemia. We have investigated the molecular basis of the Lutheran:Laminin 10/11 interaction. Lutheran binding to Laminin 10/11 is pH and salt dependent suggesting the interaction is influenced by surface charge. Since Laminins are known to contain areas of positive charge that are of importance in binding to other ligands (heparin, alpha-dystroglycan), a molecular model of Lutheran glycoprotein was constructed to identify surface exposed areas of negatively charged aspartic and glutamic acid residues. Selected residues were mutated to alanine and the mutant proteins examined for binding to Laminin 10/11 using ELISA and Surface Plasmon Resonance. Mutations E309A and D310A greatly reduced binding to Laminin 10/11 while D312A completely abolished binding. The Lutheran model predicts a rod-like structure with a flexible hinge region of 6–8 residues between the 2nd and 3rd IgSF domains. Residues E309, D310 and D312 are located on domain 3 proximal to the hinge region. Mutations (H235P, and delta 233–235) within the hinge region also abolished Laminin binding showing the hinge region to be essential for ligand interaction. Electron tomography on recombinant Lutheran-Fc chimeric protein bound to Laminin 10/11 suggested Lutheran glycoprotein bends at the hinge region to expose the critical negatively charged residues on domain 3 and thereby allow Laminin binding. These data suggest Lutheran-Laminin 10/11 interaction is a novel type of protein:protein interaction and provide a foundation for further investigation of its biological significance.
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Su, Ya-Chi, Dipali Sinha, and Peter N. Walsh. "Localization of Ligand-Binding Exosites In the Catalytic Domain of Factor XIa." Blood 116, no. 21 (November 19, 2010): 1148. http://dx.doi.org/10.1182/blood.v116.21.1148.1148.
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Abstract Abstract 1148 Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, and FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed experiments showing that the proteolytic activation of FIX by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXIa-R5 and FXIa-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity using a kallikrein-specific substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 3% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen by FXIa-R4 were similar to those achieved by kallikrein but not by FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. From the co-crystal structure of the FXIa catalytic domain with PN2KPI, the residues R3704, Y5901, E98, Y143, I151, and K192 (chymotrypsin numbering) in the FXIa catalytic domain have been identified to be possibly involved in the interactions with its inhibitors. A single mutation comprising Y5901A in the R2 region of FXIa does not affect folding however this mutant displayed resistance to inhibition by PN2KPI indicating that Y5901 is involved in the interaction of FXIa with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa in the interaction of FXIa with PN2KPI. Disclosures: No relevant conflicts of interest to declare.
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Solowska, J., J. M. Edelman, S. M. Albelda, and C. A. Buck. "Cytoplasmic and transmembrane domains of integrin beta 1 and beta 3 subunits are functionally interchangeable." Journal of Cell Biology 114, no. 5 (September 1, 1991): 1079–88. http://dx.doi.org/10.1083/jcb.114.5.1079.
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Integrin beta subunits combine with specific sets of alpha subunits to form functional adhesion receptors. The structure and binding properties of integrins suggest the presence of domains controlling at least three major functions: subunit association, ligand binding, and cytoskeletal interactions. To more carefully define structure/function relationships, a cDNA construct consisting of the extracellular domain of the avian beta 1 subunit and the cytoplasmic and transmembrane domains of the human beta 3 subunit was prepared and expressed in murine 3T3 cells. The resulting chimeric beta 1/3 subunit formed heterodimers with alpha subunits from the beta 1 subfamily, could not interact with alpha IIb from the beta 3 subfamily, was targeted to focal contacts, and formed functional complexes within the focal contacts. A second cDNA construct was prepared that coded for an avian beta 1 subunit without a transmembrane or cytoplasmic domain. This subunit was not found in association with an accompanying alpha subunit, nor was it found expressed on the cell surface. Instead, it accumulated in vesicles within the cytoplasm and was eventually shed from the cell. The results from studies of the behavior of these two cDNA constructs demonstrate that the transmembrane and cytoplasmic domains play no role in alpha subunit selection, that the cytoplasmic domain of beta 3 is capable of functioning in the context of alpha subunits with which it is not normally paired, and that both integrin subunits must be membrane associated for normal assembly and transport to cell surface adhesive structures.
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Düsterhöft, Stefan, Selcan Kahveci-Türköz, Justyna Wozniak, Anke Seifert, Petr Kasparek, Henrike Ohm, Shixin Liu, et al. "The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release." Cellular and Molecular Life Sciences 78, no. 11 (May 5, 2021): 5015–40. http://dx.doi.org/10.1007/s00018-021-03845-3.
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AbstractMembrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.
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Jääskeläinen, J., A. Deeb, J. W. Schwabe, N. P. Mongan, H. Martin, and I. A. Hughes. "Human androgen receptor gene ligand-binding-domain mutations leading to disrupted interaction between the N- and C-terminal domains." Journal of Molecular Endocrinology 36, no. 2 (April 2006): 361–68. http://dx.doi.org/10.1677/jme.1.01885.
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Most mutations in the androgen receptor (AR) ligand-binding domain (LBD) disrupt binding of the natural ligands: dihydrotestosterone and testosterone. Some AR LBD mutations do not affect ligand binding but they disrupt androgen-induced interaction of the N-terminal motif FXXLF and C-terminal activation function 2 (AF2). As N-/C-terminal interaction requires binding of agonists that have androgen activity in vivo, it correlates well with the phenotype. To study this further, we searched the Cambridge intersex database for patients with a detected missense mutation in the AR LBD presenting with normal ligand binding. Six mutations (D695N, Y763C, R774H, Q798E, R855H and L907F) were selected and introduced by site-directed mutagenesis into the pSVAR and pM-LBD plasmids. The transactivational potential of the wild-type and mutant androgen receptors (pSVAR) was examined by dual-luciferase assay using pGRE-LUC as a reporter vector. N-/C-terminal interaction was studied by mammalian two-hybrid assay using wild-type and mutated AR LBD (pM-LBD), pVP16-rAR-(5–538) (encoding rat amino-terminal AR) and pCMX-UAS-TK-LUC as a reporter. AR LBD mutations D695N, R774H and L907F presented with minimal transactivational capacity and N-/C-terminal interaction was totally disrupted. Mutations Y763C and R885H had some residual dose-dependent transactivational potential and minimal N-/C-terminal interaction. Q798E presented with good transactivational potential and it showed only mild reduction in N-/C-terminal interaction. With the selected mutations, N-/C-terminal interaction correlated well with AR transactivation and the phenotype. Disrupted N-/C-terminal interaction is capable of providing the mechanism for androgen-insensitivity syndrome in most cases where the mutation in the LBD does not disrupt ligand binding. Furthermore, mutations leading to the disrupted N-/C-terminal interaction can be localized to certain critical regions in the three-dimensional structure of the AR LBD. Our study shows that apart from the previously reported regions, regions just before helix 3, between helices 5 and 6, and at helix 10 are also important for AR N-/C-terminal interaction.
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Gustafsson, Jan-Åke. "Steroids and the Scientist." Molecular Endocrinology 19, no. 6 (June 1, 2005): 1412–17. http://dx.doi.org/10.1210/me.2004-0479.
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Abstract Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor α); 2) liver X receptor β (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor β, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor β agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.
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Mukhopadhyay, Subhankar, Audrey Varin, Yunying Chen, Baoying Liu, Karl Tryggvason, and Siamon Gordon. "SR-A/MARCO–mediated ligand delivery enhances intracellular TLR and NLR function, but ligand scavenging from cell surface limits TLR4 response to pathogens." Blood 117, no. 4 (January 27, 2011): 1319–28. http://dx.doi.org/10.1182/blood-2010-03-276733.
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Abstract Phagocytic and pathogen sensing receptors are responsible for particle uptake and inflammation. It is unclear how these receptors' systems influence each other's function to shape an innate response. The class-A scavenger receptors SR-A (scavenger receptor A) and MARCO (macrophage receptor with collagenous structure) are 2 well-characterized phagocytic receptors that are unable to initiate inflammatory responses by themselves, yet are implicated in the pathogenesis of various inflammatory disorders. However, the mechanism for such an apparent discrepancy is still unclear. We utilized SR-A−/−, MARCO−/−, and SR-A−/−-MARCO−/− mice, along with microbe-derived, environmental, and synthetic polyanions to assess the inflammatory responses following combinatorial ligation of SR-A/MARCO and selected Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) by their shared ligands. In addition to ligating SR-A and MARCO, these agonists also selectively activated the cell-surface sensor TLR4, endosomal TLR3, and the cytosolic NOD2 and NALP3 (NACHT domain–, leucine-rich repeat–, and pyrin domain–containing protein 3). We show that, following recognition of common ligands, SR-A and MARCO attenuate TLR4-mediated responses while enhancing responses by the intracellular TLR3, NOD2, and NALP3. We conclude that SR-A/MARCO-mediated rapid ligand internalization prevented sensing by surface TLRs while increasing ligand availability in intracellular compartments, thus allowing sensing and robust responses by intracellular sensors.
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Nowicki, B., A. Hart, K. E. Coyne, D. M. Lublin, and S. Nowicki. "Short consensus repeat-3 domain of recombinant decay-accelerating factor is recognized by Escherichia coli recombinant Dr adhesin in a model of a cell-cell interaction." Journal of Experimental Medicine 178, no. 6 (December 1, 1993): 2115–21. http://dx.doi.org/10.1084/jem.178.6.2115.
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A bacterial pathogen that is important in both urinary tract and intestinal infections is Escherichia coli which expresses Dr or related adhesins. In this report, we present a model for testing cell-cell interaction, using both molecularly characterized laboratory cells that express recombinant molecules of human decay-accelerating factor (DAF), and recombinant bacterial Dr colonization factors. Dr adhesin ligand was identified as DAF (CD55), a membrane protein that protects autologous tissues from damage due to the complement system. Structure-function studies mapped the adhesin-binding site on the DAF molecule. A single-point substitution in the third short consensus repeat domain, Ser165 to Leu, corresponding to the Dra to Drb allelic polymorphism, caused complete abolition of adhesin binding to DAF.
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Hung, Tzu-Chieh, Tung-Ti Chang, Ming-Jen Fan, Cheng-Chun Lee, and Calvin Yu-Chian Chen. "In SilicoInsight into Potent of Anthocyanin Regulation of FKBP52 to Prevent Alzheimer’s Disease." Evidence-Based Complementary and Alternative Medicine 2014 (2014): 1–20. http://dx.doi.org/10.1155/2014/450592.
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Alzheimer’s disease (AD) is caused by the hyperphosphorylation of Tau protein aggregation. FKBP52 (FK506 binding protein 52) has been found to inhibit Tau protein aggregation. This study found six different kinds of anthocyanins that have high binding potential. After analyzing the docking positions, hydrophobic interactions, and hydrogen bond interactions, several amino acids were identified that play important roles in protein and ligand interaction. The proteins’ variation is described using eigenvectors and the distance between the amino acids during a molecular dynamics simulation (MD). This study investigates the three loops based around Glu85, Tyr113, and Lys121—all of which are important in inducing FKBP52 activation. By performing a molecular dynamic simulation process between unbound proteins and the protein complex with FK506, it was found that ligand targets that docked onto the FK1 domain will decrease the distance between Glu85/Tyr113 and Glu85/Lys121. The FKBP52 structure variation may induce FKBP52 activation and inhibit Tau protein aggregation. The results indicate that anthocyanins might change the conformation of FKBP52 during binding. In addition, the purple anthocyanins, such as cyanidin-3-glucoside and malvidin-3-glucoside, might be better than FK506 in regulating FKBP52 and treating Alzheimer’s disease.
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Forgac, M. "Structure, function and regulation of the coated vesicle V-ATPase." Journal of Experimental Biology 172, no. 1 (November 1, 1992): 155–69. http://dx.doi.org/10.1242/jeb.172.1.155.
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The coated vesicle V-ATPase plays an important role in both receptor-mediated endocytosis and intracellular membrane traffic by providing the acidic environment required for ligand-receptor dissociation and receptor recycling. The coated vesicle V-ATPase is a macromolecular complex of relative molecular mass 750,000 composed of nine subunits arranged in two structural domains. The peripheral V1 domain, which has a relative molecular mass of 500,000, has the subunit structure 73(3)58(3)40(1)34(1)33(1) and possesses all the nucleotide binding sites of the V-ATPase. The integral Vo domain of relative molecular mass 250,000 has a subunit composition of 100(1)38(1)19(1)17(6) and possesses the pathway for proton conduction across the membrane. Reassembly studies have allowed us to probe the role of specific subunits in the V-ATPase complex while chemical labeling studies have allowed us to identify specific residues which play a critical role in catalysis. From both structural analysis and sequence homology, the vacuolar-type H(+)-ATPases resemble the F-type H(+)-ATPases. Unlike the F1 and Fo domains of the F-type ATPases, however, the V1 and Vo domains do not appear to function independently. The possible relevance of these observations to the regulation of vacuolar acidification is discussed.
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Hahn, W. C., and B. E. Bierer. "Separable portions of the CD2 cytoplasmic domain involved in signaling and ligand avidity regulation." Journal of Experimental Medicine 178, no. 5 (November 1, 1993): 1831–36. http://dx.doi.org/10.1084/jem.178.5.1831.
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Effective T cell immune responses require the molecular interplay between adhesive and signaling events mediated by the T cell receptor for antigen (TCR) and other cell surface coreceptor molecules. In this report, we have distinguished between the role of regulated adhesion and transmembrane signaling in coreceptor function using the T cell glycoprotein CD2. By binding its ligands on antigen-presenting cell (APC), CD2 serves both to initiate signal transduction events and to promote cellular adhesion. Furthermore, the avidity of CD2 for one ligand, CD58 (LFA-3), is regulated by TCR signaling. We have expressed wild type CD2 and a series of mutated CD2 molecules in an antigen-specific murine T cell hybridoma. Structure-function studies using these stably transfected cell lines identify two structurally and functionally distinct regions of the 116 amino acid (aa) cytoplasmic domain. One region is required for CD2-mediated signal transduction, and a separate COOH-terminal 21 aa portion is required for CD2 activity regulation. Cell lines expressing CD2 molecules lacking the cytoplasmic segment required for CD2-initiated IL-2 production retain the ability to upregulate CD2 avidity. Conversely, cell lines expressing CD2 mutants lacking the cytoplasmic segment required for avidity regulation retain the ability to initiate CD2-specific signaling. In antigen-specific T cell responses, basal binding of CD2 to its ligands enhances antigen responsiveness only minimally, whereas regulated avidity and transmembrane signaling are both required for optimal coreceptor function. Taken together, these studies demonstrate the independent contributions of regulated adhesion and intracellular signaling in CD2 coreceptor function.
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Chuntharapai, A., V. Gibbs, J. Lu, A. Ow, S. Marsters, A. Ashkenazi, A. De Vos, and K. Jin Kim. "Determination of Residues Involved in Ligand Binding and Signal Transmission in the Human IFN-α Receptor 2." Journal of Immunology 163, no. 2 (July 15, 1999): 766–73. http://dx.doi.org/10.4049/jimmunol.163.2.766.
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Abstract The human IFN-α receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-α2/α1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-α2/α1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-α8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-α2, but remained responsive to hIFN-β. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-α2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.