Structural basis for two efficient modes of agropinic acid opine import into the bacterial pathogen Agrobacterium tumefaciens

2019 ◽  
Vol 476 (1) ◽  
pp. 165-178 ◽  
Author(s):  
Loïc Marty ◽  
Armelle Vigouroux ◽  
Magali Aumont-Nicaise ◽  
Franck Pelissier ◽  
Thibault Meyer ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Time Course ◽  
Genetically Modify ◽  
Bacterial Pathogen ◽  
Binding Mode ◽  
Structural Basis ◽  
Crystallization Time ◽  
Periplasmic Binding Proteins ◽  
Plant Tumors ◽  
Nanomolar Range

AbstractAgrobacterium tumefaciens pathogens genetically modify their host plants to drive the synthesis of opines in plant tumors. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. These opines serve as nutrients and are imported into bacteria via periplasmic-binding proteins (PBPs) in association with ABC transporters. Structural and affinity data on agropine and agropinic acid opines bound to PBPs are currently lacking. Here, we investigated the molecular basis of AgtB and AgaA, proposed as the specific PBP for agropine and agropinic acid import, respectively. Using genetic approaches and affinity measurements, we identified AgtB and its transporter as responsible for agropine uptake in agropine-assimilating agrobacteria. Nonetheless, we showed that AgtB binds agropinic acid with a higher affinity than agropine, and we structurally characterized the agropinic acid-binding mode through three crystal structures at 1.4, 1.74 and 1.9 Å resolution. In the crystallization time course, obtaining a crystal structure of AgtB with agropine was unsuccessful due to the spontaneous lactamization of agropine into agropinic acid. AgaA binds agropinic acid only with a similar affinity in nanomolar range as AgtB. The structure of AgaA bound to agropinic acid at 1.65 Å resolution defines a different agropinic acid-binding signature. Our work highlights the structural and functional characteristics of two efficient agropinic acid assimilation pathways, of which one is also involved in agropine assimilation.

scholarly journals The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum

2018 ◽  
Vol 293 (21) ◽  
pp. 7930-7941 ◽  
Author(s):  
Thibault Meyer ◽  
Armelle Vigouroux ◽  
Magali Aumont-Nicaise ◽  
Gilles Comte ◽  
Ludovic Vial ◽  
...  
Keyword(s):  
Competitive Advantage ◽  
Plant Defense ◽  
Higher Plants ◽  
Bacterial Pathogen ◽  
Binding Mode ◽  
Plant Colonization ◽  
Plant Metabolites ◽  
Tomato Plants ◽  
Periplasmic Binding Proteins ◽  
Nanomolar Range

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose–containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.

scholarly journals Import pathways of the mannityl-opines into the bacterial pathogen Agrobacterium tumefaciens: structural, affinity and in vivo approaches

2020 ◽  
Vol 477 (3) ◽  
pp. 615-628
Author(s):  
Armelle Vigouroux ◽  
Jeanne Doré ◽  
Loïc Marty ◽  
Magali Aumont-Nicaise ◽  
Pierre Legrand ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Tumor Development ◽  
Bacterial Pathogen ◽  
Binding Mode ◽  
Acid Uptake ◽  
Crystal Forms ◽  
Plant Tumor ◽  
Structural Affinity

Agrobacterium tumefaciens pathogens use specific compounds denoted opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ATP-binding cassette transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Structural and affinity data on mannopinic acid bound to SBPs are currently lacking while those of the three others mannityl opines are available. We investigated the molecular basis of two pathways for mannopinic acid uptake. MoaA was proposed as the specific SBP for mannopinic acid import in mannityl opines-assimilating agrobacteria, which was validated here using genetic studies and affinity measurements. We structurally characterized the mannopinic acid-binding mode of MoaA in two crystal forms at 2.05 and 1.57 Å resolution. We demonstrated that the non-specific SBP MotA, so far characterized as mannopine and Amadori compound importer, was also able to transport mannopinic acid. The structure of MotA bound to mannopinic acid at 2.2 Å resolution defines a different mannopinic acid-binding signature, similar to that of mannopine. Combining in vitro and in vivo approaches, this work allowed us to complete the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.

scholarly journals Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

2021 ◽  
Vol 7 (5) ◽  
pp. eabc6479
Author(s):  
Sarah A. Mosure ◽  
Timothy S. Strutzenberg ◽  
Jinsai Shang ◽  
Paola Munoz-Tello ◽  
Laura A. Solt ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Binding Mode ◽  
Structural Basis ◽  
Receptor Interaction ◽  
Heme Binding ◽  
Endogenous Ligand ◽  
Interaction Domain ◽  
Response Elements ◽  
Biochemical Assays ◽  
Chemical Cross Linking

Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. Cellular studies indicate that heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription. However, fluorescence-based biochemical assays suggest that heme displaces NCoR; here, we show that this is due to a heme-dependent artifact. Using ITC and NMR spectroscopy, we show that heme binding remodels the thermodynamic interaction profile of NCoR receptor interaction domain (RID) binding to REV-ERBβ ligand-binding domain (LBD). We solved two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, revealing the heme-dependent NCoR binding mode. ITC and chemical cross-linking mass spectrometry reveals a 2:1 LBD:RID stoichiometry, consistent with cellular studies showing that NCoR-dependent repression of REV-ERB transcription occurs on dimeric DNA response elements. Our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

scholarly journals Structure activity relationships and the binding mode of quinolinone-pyrimidine hybrids as reversal agents of multidrug resistance mediated by P-gp

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jerónimo Laiolo ◽  
Priscila Ailin Lanza ◽  
Oscar Parravicini ◽  
Cecilia Barbieri ◽  
Daniel Insuasty ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Molecular Complexes ◽  
Binding Mode ◽  
Reversal Agents ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Doxorubicin Toxicity ◽  
Key Residues ◽  
Nanomolar Range ◽  
Experimental Structure

AbstractP-gp-associated multidrug resistance is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure–activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone to the moiety bearing the pyrimidine favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4–7 and 12 is required for inhibition.

scholarly journals Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

2017 ◽  
Vol 114 (33) ◽  
pp. E6942-E6951 ◽  
Author(s):  
Genevieve E. Lind ◽  
Tung-Chung Mou ◽  
Lucia Tamborini ◽  
Martin G. Pomper ◽  
Carlo De Micheli ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Binding Affinity ◽  
Binding Mode ◽  
Structural Basis ◽  
Nmda Receptor Antagonists ◽  
Receptor Antagonists ◽  
Glutamate Binding ◽  
Subunit Interface ◽  
The Central Nervous System ◽  
Contact Residues

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

scholarly journals Transcriptomic Analysis of Arabidopsis Seedlings in Response to an Agrobacterium-Mediated Transformation Process

2018 ◽  
Vol 31 (4) ◽  
pp. 445-459 ◽  
Author(s):  
Kaixuan Duan ◽  
Christopher J. Willig ◽  
Joann R. De Tar ◽  
William G. Spollen ◽  
Zhanyuan J. Zhang
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Defense Response ◽  
Time Course ◽  
Basic Research ◽  
Defense Responses ◽  
Transformation Process ◽  
Host Responses ◽  
Nucleic Acid Metabolism ◽  
Agrobacterium Strain ◽  
Agrobacterium Mediated Transformation

Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease. This pathogen is capable of transferring the T-DNA from its Ti plasmid to the host cell and, then, integrating it into the host genome. To date, this genetic transformation ability has been harnessed as the dominant technology to produce genetically modified plants for both basic research and crop biotechnological applications. However, little is known about the interaction between Agrobacterium tumefaciens and host plants, especially the host responses to Agrobacterium infection and its associated factors. We employed RNA-seq to follow the time course of gene expression in Arabidopsis seedlings infected with either an avirulent or a virulent Agrobacterium strain. Gene Ontology analysis indicated many biological processes were involved in the Agrobacterium-mediated transformation process, including hormone signaling, defense response, cellular biosynthesis, and nucleic acid metabolism. RNAseq and quantitative reverse transcription-polymerase chain reaction results indicated that expression of genes involved in host plant growth and development were repressed but those involved in defense response were induced by Agrobacterium tumefaciens. Further analysis of the responses of transgenic Arabidopsis lines constitutively expressing either the VirE2 or VirE3 protein suggested Vir proteins act to enhance plant defense responses in addition to their known roles facilitating T-DNA transformation.

Quinolin-2-one-pyrimidine Hybrids Acting as Potent Reversal Agents on the P-gp-mediated Multidrug Resistance: Insights into Structure-activity Relationships and the Binding Mode

2021 ◽  
Author(s):  
Jerónimo Laiolo ◽  
Priscila Ailin Lanza ◽  
Oscar Parravicini ◽  
Cecilia Barbieri ◽  
Daniel Insuasty ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Molecular Complexes ◽  
Binding Mode ◽  
Reversal Agents ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Doxorubicin Toxicity ◽  
Key Residues ◽  
Nanomolar Range ◽  
Experimental Structure

Abstract P-gp-associated multidrug resistance (MDR) is a major impediment to the success of chemotherapy. With the aim of finding non-toxic and effective P-gp inhibitors, we investigated a panel of quinolin-2-one-pyrimidine hybrids. Among the active compounds, two of them significantly increased intracellular doxorubicin and rhodamine 123 accumulation by inhibiting the efflux mediated by P-gp and restored doxorubicin toxicity at nanomolar range. Structure-activity relationships showed that the number of methoxy groups, an optimal length of the molecule in its extended conformation, and at least one flexible methylene group bridging the quinolinone moiety favored the inhibitory potency of P-gp. The best compounds showed a similar binding pattern and interactions to those of doxorubicin and tariquidar, as revealed by MD and hybrid QM/MM simulations performed with the recent experimental structure of P-gp co-crystallized with paclitaxel. Analysis of the molecular interactions stabilizing the different molecular complexes determined by MD and QTAIM showed that binding to key residues from TMH 4–7 and 12 is required for inhibition.

scholarly journals Inactivation Gating of Kv4 Potassium Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 641-660 ◽  
Author(s):  
Henry H. Jerng ◽  
Mohammad Shahidullah ◽  
Manuel Covarrubias
Keyword(s):  
Time Course ◽  
Membrane Potentials ◽  
Structural Basis ◽  
K Channel ◽  
Inactivation Curve ◽  
Double Mutation ◽  
K Channels ◽  
Closed State ◽  
Recovery From Inactivation ◽  
Inactivation Gating

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174– 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163–174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603–626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316–2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance–voltage curve (∼5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specifc residue in the S4–S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404,406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

scholarly journals Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist

2020 ◽  
Author(s):  
Polina Isaikina ◽  
Ching-Ju Tsai ◽  
Nikolaus Dietz ◽  
Filip Pamula ◽  
Anne Grahl ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Binding Mode ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Cc Chemokine ◽  
Gi Protein ◽  
Agonist And Antagonist ◽  
Cc Chemokine Receptor 5 ◽  
Chemokine Receptor 5

AbstractThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in the pathology of cancer, HIV, and COVID-19. Despite its significance as a drug target, the activation mechanism of CCR5, i.e. how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N-terminus of agonist chemokines pushes onto an aromatic connector that transmits activation to the canonical GPCR microswitch network. This activation mechanism differs significantly from other CC chemokine receptors that bind shorter chemokines in a shallow binding mode and have unique sequence signatures and a specialized activation mechanism.One-sentence summaryThe structure of CCR5 in complex with the chemokine agonist [6P4]CCL5 and the heterotrimeric Gi protein reveals its activation mechanism

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