scholarly journals The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3513-3520 ◽  
Author(s):  
Vineet Gupta ◽  
Annette Gylling ◽  
José Luis Alonso ◽  
Takashi Sugimori ◽  
Petre Ianakiev ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Divalent Cations ◽  
Contact Region ◽  
Allosteric Modulator ◽  
Tail Domain ◽  
Wild Type ◽  
High Affinity

Abstract Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

A Glycan Wedge Between βTD and βI Domains Activates Integrin αIIbβ3,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3259-3259
Author(s):  
Jun Yamanouchi ◽  
Takaaki Hato ◽  
Hiroshi Fujiwara ◽  
Yoshihiro Yakushijin ◽  
Masaki Yasukawa
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Conflicts Of Interest ◽  
Consensus Sequence ◽  
Glycosylation Site ◽  
Tail Domain ◽  
Wild Type ◽  
Integrin Activation ◽  
Integrin Αiibβ3 ◽  
Constitutively Active

Abstract Abstract 3259 Integrin αIIbβ3 undergoes allosteric conformational changes in its extracellular domains, resulting in integrin activation that allows high affinity binding with soluble ligands. The crystal structure of the integrin β subunit revealed an interaction of the β-tail domain (βTD) with the βI domain containing ligand-binding sites, suggesting that βTD may be involved in allosteric mechanism for integrin activation. However, previous studies have shown conflicting results on the functional role of βTD in integrin activation. In this study, we conducted site-directed mutagenesis in the βTD domain and tested ligand binding to αIIbβ3 mutants. We produced αIIbβ3 mutants in which the β3TD loop residues (DSSG) were substituted with the corresponding β1 (NGNN) or β2TD residues (DGMD). The αIIbβ3 mutants were expressed on the surface of CHO cells by cotransfection of mutant β3 and wild-type αIIb cDNAs, and were tested for binding of PAC1, a ligand-mimetic anti-αIIbβ3 antibody. The NGNN, but not DGMD mutant bound significant PAC1 binding without any stimulation, indicating a constitutively active state. To identify the residue(s) responsible for αIIbβ3 activation in the βTD, we produced αIIbβ3 mutants in which the individual residues in the β3TD loop were substituted with the corresponding β1TD residues. Among them, only G675N bound significant PAC1 binding without any stimulation. Since G675N mutation creates a sequence known to be a consensus sequence for glycosylation (Asn-X-Ser/Thr), it is possible that the insertion of glycans into the βTD loop induces conformational changes in αIIbβ3 which allow ligand binding. To test this hypothesis, we added substitution of S677 with Thr, Ala or Asp to the G675N mutation. The resultant G675N/S677T double mutant, in which the N-glycosylation site was preserved, was constitutively active. In contrast, G675N/S677A and G675N/S677D, in which the N-glycosylation site was disrupted, were in an inactive state. These results suggest that an artificial glycan wedge between βTD and βI domains activates αIIbβ3. However, our study does not provide evidence that the βTD domain constrains wild type αIIbβ3 inactive although the separation of βTD and βI domains may be able to activate integrins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

2021 ◽  
Author(s):  
Amit Ketkar ◽  
Lane Smith ◽  
Callie Johnson ◽  
Alyssa Richey ◽  
Makayla Berry ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutation Frequency ◽  
Control Sequence ◽  
Wild Type ◽  
Binding Properties ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex ◽  
Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

scholarly journals Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1)

2015 ◽  
Vol 308 (8) ◽  
pp. C631-C641 ◽  
Author(s):  
Michele Visentin ◽  
Ersin Selcuk Unal ◽  
Mitra Najmi ◽  
Andras Fiser ◽  
Rongbao Zhao ◽  
...  
Keyword(s):  
Choroid Plexus ◽  
Rate Constant ◽  
Binding Pocket ◽  
Wild Type ◽  
Efflux Rate ◽  
High Affinity ◽  
Extracellular Compartment ◽  
Efflux Rate Constant ◽  
Opposite Compartment

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/ Ki for [3H]methotrexate and [3H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [3H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier's mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.

scholarly journals Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions

2007 ◽  
Vol 81 (15) ◽  
pp. 7833-7843 ◽  
Author(s):  
Joshua C. Grieger ◽  
Jarrod S. Johnson ◽  
Brittney Gurda-Whitaker ◽  
Mavis Agbandje-McKenna ◽  
R. Jude Samulski
Keyword(s):  
Conformational Changes ◽  
Structural Changes ◽  
Wild Type ◽  
Dot Blot ◽  
Adeno Associated Virus ◽  
N Terminus ◽  
Localization Signal ◽  
Significant Effort

ABSTRACT Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.

scholarly journals The role of water and protein flexibility in the structure-based virtual screening of allosteric GPCR modulators: an mGlu5 receptor case study

2019 ◽  
Vol 33 (9) ◽  
pp. 787-797 ◽  
Author(s):  
Zoltán Orgován ◽  
György G. Ferenczy ◽  
György M. Keserű
Keyword(s):  
Virtual Screening ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Metabotropic Glutamate Receptor ◽  
Protein Structures ◽  
Protein Flexibility ◽  
Water Molecules ◽  
Large Set ◽  
Allosteric Modulators

Abstract Stabilizing unique receptor conformations, allosteric modulators of G-protein coupled receptors (GPCRs) might open novel treatment options due to their new pharmacological action, their enhanced specificity and selectivity in both binding and signaling. Ligand binding occurs at intrahelical allosteric sites and involves significant induced fit effects that include conformational changes in the local protein environment and water networks. Based on the analysis of available crystal structures of metabotropic glutamate receptor 5 (mGlu5) we investigated these effects in the binding of mGlu5 receptor negative allosteric modulators. A large set of retrospective virtual screens revealed that the use of multiple protein structures and the inclusion of selected water molecules improves virtual screening performance compared to conventional docking strategies. The role of water molecules and protein flexibility in ligand binding can be taken into account efficiently by the proposed docking protocol that provided reasonable enrichment of true positives. This protocol is expected to be useful also for identifying intrahelical allosteric modulators for other GPCR targets.

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

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

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

scholarly journals Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova

Reproduction ◽  
2012 ◽  
Vol 143 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Sayaka Koyanagi ◽  
Hiroko Hamasaki ◽  
Satoshi Sekiguchi ◽  
Kenshiro Hara ◽  
Yoshiyuki Ishii ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Proteomic Analysis ◽  
Ubiquitin Proteasome System ◽  
Underlying Mechanism ◽  
Wild Type ◽  
Transmission Electron ◽  
Ubiquitin Proteasome

Maternal proteins are rapidly degraded by the ubiquitin–proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficientgad. Furthermore, we assessed morphological features ingadmouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the ‘maternal antigen that embryos require’ (NLRP5 (MATER)) protein level increased significantly ingadmouse ova compared with that in wild-type mice. In an ultrastructural study,gadmouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

scholarly journals Biosynthesis of the Glycolipid Anchor in Lipoteichoic Acid of Staphylococcus aureus RN4220: Role of YpfP, the Diglucosyldiacylglycerol Synthase

2001 ◽  
Vol 183 (11) ◽  
pp. 3506-3514 ◽  
Author(s):  
Michael Y. Kiriukhin ◽  
Dmitri V. Debabov ◽  
Dean L. Shinabarger ◽  
Francis C. Neuhaus
Keyword(s):  
Electron Microscopy ◽  
Staphylococcus Aureus ◽  
Doubling Time ◽  
Lipoteichoic Acid ◽  
Luria Bertani ◽  
Wild Type ◽  
Glycolipid Anchor ◽  
Triton X ◽  
Pleomorphic Cells

ABSTRACT In Staphylococcus aureus RN4220, lipoteichoic acid (LTA) is anchored in the membrane by a diglucosyldiacylglycerol moiety. The gene (ypfP) which encodes diglucosyldiacylglycerol synthase was recently cloned from Bacillus subtilis and expressed in Escherichia coli (P. Jorasch, F. P. Wolter, U. Zahringer, and E. Heinz, Mol. Microbiol. 29:419–430, 1998). To define the role of ypfP in this strain of S. aureus, a fragment of ypfP truncated from both ends was cloned into the thermosensitive replicon pVE6007 and used to inactivate ypfP. Chloramphenicol-resistant (ypfP::cat) clones did not synthesize the glycolipids monoglucosyldiacylglycerol and diglucosyldiacylglycerol. Thus, YpfP would appear to be the only diglucosyldiacylglycerol synthase in S. aureus providing glycolipid for LTA assembly. In LTA from the mutant, the glycolipid anchor is replaced by diacylglycerol. Although the doubling time of the mutant was identical to that of the wild type in Luria-Bertani (LB) medium, growth of the mutant in LB medium containing 1% glycine was not observed. This inhibition was antagonized by either l- or d-alanine. Moreover, viability of the mutant at 37°C in 0.05 M phosphate (pH 7.2)-saline for 12 h was reduced to <0.1%. Addition of 0.1% d-glucose to the phosphate-saline ensured viability under these conditions. The autolysis of the ypfP::cat mutant in the presence of 0.05% Triton X-100 was 1.8-fold faster than that of the parental strain. Electron microscopy of the mutant revealed not only a small increase in cell size but also the presence of pleomorphic cells. Each of these phenotypes may be correlated with either (or both) a deficiency of free glycolipid in the membrane or the replacement of the usual glycolipid anchor of LTA with diacylglycerol.

scholarly journals Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Rna Seq ◽  
Wild Type ◽  
Content Type ◽  
High Affinity ◽  
Polyphosphate Metabolism

ABSTRACT Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

Role of Dectin-2 in the phagocytosis of Cryptococcus neoformans by dendritic cells

2021 ◽  
Author(s):  
Yuki Kitai ◽  
Ko Sato ◽  
Daiki Tanno ◽  
Xiaoliang Yuan ◽  
Aya Umeki ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Cryptococcus Neoformans ◽  
Conformational Changes ◽  
Cell Walls ◽  
Actin Polymerization ◽  
Similar Degree ◽  
Wild Type ◽  
Lectin Receptor ◽  
Adapter Molecule

The cell walls and capsules of Cryptococcus neoformans , a yeast-type fungal pathogen, are rich in polysaccharides. Dectin-2 is a C-type lectin receptor (CLR) that recognizes high-mannose polysaccharides. Previously, we demonstrated that Dectin-2 is involved in cytokine production by bone marrow-derived dendritic cells (BM-DCs) in response to stimulation with C. neoformans . In the present study, we analyzed the role of Dectin-2 in the phagocytosis of C. neoformans by BM-DCs. The engulfment of this fungus by BM-DCs was significantly decreased in mice lacking Dectin-2 (Dectin-2KO) or caspase recruitment domain-containing protein 9 (CARD9KO), a common adapter molecule that delivers signals triggered by CLRs, compared to wild-type (WT) mice. Phagocytosis was likewise inhibited, to a similar degree, by the inhibition of Syk, a signaling molecule involved in CLR-triggered activation. A PI3K inhibitor, in contrast, completely abrogated the phagocytosis of C. neoformans . Actin polymerization, i.e., conformational changes in cytoskeletons detected at sites of contact with C. neoformans , was also decreased in BM-DCs of Dectin-2KO and CARD9KO mice. Finally, the engulfment of C. neoformans by macrophages was significantly decreased in the lungs of Dectin-2KO mice compared to WT mice. These results suggest that Dectin-2 may play an important role in the actin polymerization and phagocytosis of C. neoformans by DCs, possibly through signaling via CARD9 and a signaling pathway mediated by Syk and PI3K.

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