Biochemical and structural study ofArabidopsishexokinase 1

2015 ◽  
Vol 71 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Juan Feng ◽  
Shun Zhao ◽  
Xuemin Chen ◽  
Wenda Wang ◽  
Wei Dong ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Glucose Sensing ◽  
Wild Type ◽  
Structural Explanation ◽  
High Affinity ◽  
Physiological Processes ◽  
Glucose Binding ◽  
Glucose Signalling ◽  
Dual Functions ◽  
Resolution Structure

Hexokinase 1 fromArabidopsis thaliana(AtHXK1) plays a dual role in glycolysis and sugar sensing for vital metabolic and physiological processes. The uncoupling of glucose signalling from glucose metabolism was demonstrated by the analysis of two mutants (AtHXK1G104DandAtHXK1S177A) that are catalytically inactive but still functional in signalling. In this study, substrate-binding experiments indicate that the two catalytically inactive mutants have a high affinity for glucose, and an ordered substrate-binding mechanism has been observed for wild-typeAtHXK1. The structure ofAtHXK1 was determined both in its inactive unliganded form and in its active glucose-bound form at resolutions of 1.8 and 2.0 Å, respectively. These structures reveal a domain rearrangement ofAtHXK1 upon glucose binding. The 2.1 Å resolution structure ofAtHXK1S177Ain the glucose-bound form shows similar glucose-binding interactions as the wild type. A glucose-sensing network has been proposed based on these structures. Taken together, the results provide a structural explanation for the dual functions ofAtHXK1.

scholarly journals Evidence of Evolutionary Conservation of Function between the Thyroxine Transporter Oatp1c1 and Major Facilitator Superfamily Members

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5941-5951 ◽  
Author(s):  
Daniel E. Westholm ◽  
Jacob D. Marold ◽  
Kevin J. Viken ◽  
Alicia H. Duerst ◽  
Grant W. Anderson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Substrate Binding ◽  
Evolutionary Conservation ◽  
Major Facilitator Superfamily ◽  
Transport Activity ◽  
Wild Type ◽  
Michaelis Constant ◽  
High Affinity ◽  
Major Facilitator

Organic anion transporting polypeptide 1c1 (Oatp1c1) is a high-affinity T4 transporter expressed in brain barrier cells. To identify Oatp1c1 amino acid residues critical for T4 transport, consensus membrane topology was predicted and a three-dimensional Oatp1c1 structure was generated using the known structures of major facilitator superfamily (MFS) transporters, glycerol 3-phosphate transporter, lactose permease, and the multidrug transporter Escherichia coli multidrug resistance protein D as templates. A total of nine amino acid mutations were generated based on amino acid conservation, localization to putative transmembrane domains, and side chain functionality. Mutant constructs were transiently transfected into human embryonic kidney 293 cells and assessed for plasma membrane localization and the capacity to transport substrate 125I-T4. Wild-type Oatp1c1, R601S, P609A, W277A/W278A, W277F/W278F, G399A/G409A, and G399L/G409L were all expressed at the plasma membrane. Wild-type Oatp1c1 and W277F/W278F displayed biphasic T4 transport kinetics, albeit the mutant did so with an approximately 10-fold increase in high-affinity Michaelis constant. The W277A/W278A mutation abolished Oatp1c1 T4 transport. G399A/G409A and G399V/G409V mutants displayed near wild-type activity in an uptake screen but exhibited diminished T4 transport activity at high-substrate concentrations, suggesting a substrate binding site collapse or inability to convert between input and output states. Finally, transmembrane domain 11 mutants R601S and P609A displayed partial T4 transport activity with significantly reduced maximum velocities and higher Michaelis constant. Arg601 is functionally strongly conserved with members of the MFS whose structures and function have been extensively studied. These data provide the experimental foundation for mapping Oatp1c1 substrate binding sites and reveal evolutionary conservation with bacterial MFS transporter members.

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 Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

2003 ◽  
Vol 122 (3) ◽  
pp. 295-306 ◽  
Author(s):  
Sonia Traverso ◽  
Laura Elia ◽  
Michael Pusch
Keyword(s):  
Chloride Channels ◽  
Bound State ◽  
Side Chain ◽  
Pore Channel ◽  
Kinetic Properties ◽  
Wild Type ◽  
High Affinity ◽  
Critical Residue ◽  
Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

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.

Structural studies of complexes of kallikrein 4 with wild-type and mutated forms of the Kunitz-type inhibitor BbKI

2021 ◽  
Vol 77 (8) ◽  
Author(s):  
Mi Li ◽  
Jaroslav Srp ◽  
Michael Mareš ◽  
Alexander Wlodawer ◽  
Alla Gustchina
Keyword(s):  
Serine Proteases ◽  
Structural Studies ◽  
Wild Type ◽  
Inhibitory Potency ◽  
Crystal Forms ◽  
Space Groups ◽  
Bovine Trypsin ◽  
Kallikrein 4 ◽  
Kunitz Type ◽  
Resolution Structure

Structures of BbKI, a recombinant Kunitz-type serine protease inhibitor from Bauhinia bauhinioides, complexed with human kallikrein 4 (KLK4) were determined at medium-to-high resolution in four crystal forms (space groups P3121, P6522, P21 and P61). Although the fold of the protein was virtually identical in all of the crystals, some significant differences were observed in the conformation of Arg64 of BbKI, the residue that occupies the S1 pocket in KLK4. Whereas this residue exhibited two orientations in the highest resolution structure (P3121), making either a canonical trypsin-like interaction with Asp189 of KLK4 or an alternate interaction, only a single alternate orientation was observed in the other three structures. A neighboring disulfide, Cys191–Cys220, was partially or fully broken in all KLK4 structures. Four variants of BbKI in which Arg64 was replaced by Met, Phe, Ala and Asp were expressed and crystallized, and their structures were determined in complex with KLK4. Structures of the Phe and Met variants complexed with bovine trypsin and of the Phe variant complexed with α-chymotrypsin were also determined. Although the inhibitory potency of these variant forms of BbKI was lowered by up to four orders of magnitude, only small changes were seen in the vicinity of the mutated residues. Therefore, a totality of subtle differences in KLK4–BbKI interactions within the fully extended interface in the structures of these variants might be responsible for the observed effect. Screening of the BbKI variants against a panel of serine proteases revealed an altered pattern of inhibitory specificity, which was shifted towards that of chymotrypsin-like proteases for the hydrophobic Phe and Met P1 substitutions. This work reports the first structures of plant Kunitz inhibitors with S1-family serine proteases other than trypsin, as well as new insights into the specificity of inhibition of medically relevant kallikreins.

Generation of two high affinity anti-mouse FcRn antibodies: Inhibition of IgG recycling in wild type mice and effect in a mouse model of immune thrombocytopenia

2019 ◽  
Vol 66 ◽  
pp. 362-365 ◽  
Author(s):  
Bryan Smith ◽  
Louis Christodoulou ◽  
Alison Clargo ◽  
Alison Eddleston ◽  
Kevin Greenslade ◽  
...  
Keyword(s):  
Mouse Model ◽  
Immune Thrombocytopenia ◽  
Wild Type ◽  
High Affinity

Neurotensin Enhances Locomotor Activity and Arousal and Inhibits Melanin-Concentrating Hormone Signaling

2019 ◽  
Vol 110 (1-2) ◽  
pp. 35-49 ◽  
Author(s):  
Talia Levitas-Djerbi ◽  
Dana Sagi ◽  
Ilana Lebenthal-Loinger ◽  
Tali Lerer-Goldshtein ◽  
Lior Appelbaum
Keyword(s):  
Locomotor Activity ◽  
Hormone Receptor ◽  
Behavioral Response ◽  
Hormone Signaling ◽  
Wild Type ◽  
Expression Levels ◽  
Physiological Processes ◽  
Melanin Concentrating Hormone ◽  
Behavioral Assays

Background: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. Materials and Methods: In order to study the role of Nts, nts mutant (nts–/–), and overexpressing zebrafish were generated. Results: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts–/– larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts–/– larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts–/– larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts–/– larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts–/– larvae. Conclusion: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.

XAFS study of the high-affinity copper-binding site of human PrP 91–231 and its low-resolution structure in solution 1 1Edited by I. A. Wilson

2001 ◽  
Vol 311 (3) ◽  
pp. 467-473 ◽  
Author(s):  
S.Samar Hasnain ◽  
Loretta M. Murphy ◽  
Richard W. Strange ◽  
J.Günter Grossmann ◽  
Anthony R. Clarke ◽  
...  
Keyword(s):  
Binding Site ◽  
Copper Binding ◽  
Low Resolution ◽  
High Affinity ◽  
Structure In Solution ◽  
Resolution Structure

scholarly journals Ahr and Cyp1a2 genotypes both affect susceptibility to motor deficits following gestational and lactational exposure to polychlorinated biphenyls

2017 ◽  
Author(s):  
Breann T. Colter ◽  
Helen Frances Garber ◽  
Sheila M. Fleming ◽  
Jocelyn Phillips Fowler ◽  
Gregory D. Harding ◽  
...  
Keyword(s):  
Polychlorinated Biphenyls ◽  
Learning And Memory ◽  
Animal Studies ◽  
Corn Oil ◽  
Neurological Deficits ◽  
Motor Deficits ◽  
Wild Type ◽  
Impaired Performance ◽  
High Affinity ◽  
Pcb Exposure

AbstractPolychlorinated biphenyls (PCBs) are persistent organic pollutants known to cause adverse health effects and linked to neurological deficits in both human and animal studies. Children born to exposed mothers are at highest risk of learning and memory and motor deficits. We developed a mouse model that mimics human variation in the aryl hydrocarbon receptor and cytochrome P450 1A2 (CYP1A2) to determine if genetic variation increases susceptibility to developmental PCB exposure. In our previous studies, we found that high-affinity AhrbCyp1a2(-/-) and poor-affinity AhrdCyp1a2(-/-) knockout mice were most susceptible to learning and memory deficits following developmental PCB exposure compared with AhrbCyp1a2(+/+) wild type mice (C57BL/6J strain). Our follow-up studies focused on motor deficits, because human studies have identified PCBs as a potential risk factor for Parkinson’s disease. Dams were treated with an environmentally relevant PCB mixture at gestational day 10 and postnatal day 5. We used a motor battery that included tests of nigrostriatal function as well as cerebellar function, because PCBs deplete thyroid hormone, which is essential to normal cerebellar development. There was a significant effect of PCB treatment in the rotarod test with impaired performance in all three genotypes, but decreased motor learning as well in the two Cyp1a2(-/-) knockout lines. Interestingly, we found a main effect of genotype with corn oil-treated control Cyp1a2(-/-) mice performing significantly worse than Cyp1a2(+/+) wild type mice. In contrast, we found that PCB-treated high-affinity Ahrb mice were most susceptible to disruption of nigrostriatal function with the greatest deficits in AhrbCyp1a2(-/-) mice. We conclude that differences in both genes affect susceptibility to motor deficits following developmental PCB exposure.

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