Ketamine interacts with the noradrenaline transporter at a site partly overlapping the desipramine binding site

1998 ◽  
Vol 358 (3) ◽  
pp. 328-333 ◽  
Author(s):  
Koji Hara ◽  
N. Yanagihara ◽  
Kouichiro Minami ◽  
Susumu Ueno ◽  
Yumiko Toyohira ◽  
...  
Keyword(s):  
Binding Site ◽  
Noradrenaline Transporter ◽  
Desipramine Binding ◽  
A Site

scholarly journals Autoinhibition of the formin Cappuccino in the absence of canonical autoinhibitory domains

2012 ◽  
Vol 23 (19) ◽  
pp. 3801-3813 ◽  
Author(s):  
Batbileg Bor ◽  
Christina L. Vizcarra ◽  
Martin L. Phillips ◽  
Margot E. Quinlan
Keyword(s):  
Circular Dichroism ◽  
Binding Site ◽  
Actin Polymerization ◽  
Intramolecular Interaction ◽  
Hydrodynamic Analysis ◽  
A Site ◽  
Circular Dichroïsm

Formins are a conserved family of proteins known to enhance actin polymerization. Most formins are regulated by an intramolecular interaction. The Drosophila formin, Cappuccino (Capu), was believed to be an exception. Capu does not contain conserved autoinhibitory domains and can be regulated by a second protein, Spire. We report here that Capu is, in fact, autoinhibited. The N-terminal half of Capu (Capu-NT) potently inhibits nucleation and binding to the barbed end of elongating filaments by the C-terminal half of Capu (Capu-CT). Hydrodynamic analysis indicates that Capu-NT is a dimer, similar to the N-termini of other formins. These data, combined with those from circular dichroism, suggest, however, that it is structurally distinct from previously described formin inhibitory domains. Finally, we find that Capu-NT binds to a site within Capu-CT that overlaps with the Spire-binding site, the Capu-tail. We propose models for the interaction between Spire and Capu in light of the fact that Capu can be regulated by autoinhibition.

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

Drosophila transcriptional repressor protein that binds specifically to negative control elements in fat body enhancers

1992 ◽  
Vol 12 (9) ◽  
pp. 4093-4103
Author(s):  
D Falb ◽  
T Maniatis
Keyword(s):  
Binding Site ◽  
Fat Body ◽  
Regulatory Element ◽  
Negative Control ◽  
Nuclear Extracts ◽  
Eukaryotic Proteins ◽  
Adh Gene ◽  
A Site

Expression of the Drosophila melanogaster Adh gene in adults requires a fat body-specific enhancer called the Adh adult enhancer (AAE). We have identified a protein in Drosophila nuclear extracts that binds specifically to a site within the AAE (adult enhancer factor 1 [AEF-1]). In addition, we have shown that AEF-1 binds specifically to two other Drosophila fat body enhancers. Base substitutions in the AEF-1 binding site that disrupt AEF-1 binding in vitro result in a significant increase in the level of Adh expression in vivo. Thus, the AEF-1 binding site is a negative regulatory element within the AAE. A cDNA encoding the AEF-1 protein was isolated and shown to act as a repressor of the AAE in cotransfection studies. The AEF-1 protein contains four zinc fingers and an alanine-rich sequence. The latter motif is found in other eukaryotic proteins known to be transcriptional repressors.

Identification of SAS4 and SAS5, Two Genes That Regulate Silencing in Saccharomyces cerevisiae

Genetics ◽  
1999 ◽  
Vol 153 (1) ◽  
pp. 13-23 ◽  
Author(s):  
Eugenia Y Xu ◽  
Susan Kim ◽  
Kirstin Replogle ◽  
Jasper Rine ◽  
David H Rivier
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Acute Myeloid Leukemia ◽  
Binding Site ◽  
Myeloid Leukemia ◽  
Null Alleles ◽  
Human Genes ◽  
Complementation Groups ◽  
A Site ◽  
Acute Myeloid

Abstract In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the α-mating phenotype to MATα HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

scholarly journals Identification of an octamer-binding site in the mouse kappa light-chain immunoglobulin enhancer.

1989 ◽  
Vol 9 (10) ◽  
pp. 4239-4247 ◽  
Author(s):  
R A Currie ◽  
R G Roeder
Keyword(s):  
B Cell ◽  
Binding Site ◽  
Light Chain ◽  
Specific Binding ◽  
Specific Factor ◽  
Immunoglobulin Gene ◽  
Kappa Light Chain ◽  
Nf Kappa B ◽  
Light Chain Immunoglobulin ◽  
A Site

A 215-base-pair (bp) region of the mouse MOPC 41 kappa light-chain immunoglobulin gene enhancer has been analyzed for specific binding of lymphoid and nonlymphoid nuclear factors. Mobility shift assays with a series of overlapping DNA fragments have mapped DNA-binding sites for three unique factors. The B-cell-specific (OTF-2) and ubiquitous (OTF-1) octamer-binding transcription factors specifically bound to a site centered about 136 bp 5' of the nuclear factor NF-kappa B site. A third specific factor, NF-kappa E, bound to a site that was about 75 bp 5' of the NF-kappa B site and within a region important for enhancer function. This novel factor was found in both mature B and HeLa cell nuclei. B-cell OTF-2, B-cell OTF-1, and HeLa OTF-1 bound to the kappa enhancer and kappa promoter octamer sites with similar affinities despite a 2-bp difference in the kappa enhancer octamer sequence. However, DNase I footprint analyses indicated that affinity-purified OTF-2 bound both to the enhancer OTF site and, surprisingly, to 80 bp of A + T-rich flanking sequence. Moreover, methylation interference studies demonstrated distinct differences in OTF interactions between the consensus octamer in the kappa promoter and the nonconsensus octamer identified in the enhancer. This novel observation of an OTF-binding site in the kappa enhancer provides a common link with the OTF sites in the promoter-proximal regions of all kappa promoters and thus mirrors the structural arrangement of OTF sites found in the promoters and enhancers of immunoglobulin heavy-chain genes.

Defining a second epitope for heparin-induced thrombocytopenia/thrombosis antibodies using KKO, a murine HIT-like monoclonal antibody

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1230-1236 ◽  
Author(s):  
Zhong Q. Li ◽  
Weiyi Liu ◽  
Kwang S. Park ◽  
Brue S. Sachais ◽  
Gowthani M. Arepally ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Binding Sites ◽  
Heparin Therapy ◽  
Common Complication ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
The Third ◽  
N Terminus ◽  
A Site

Heparin-induced thrombocytopenia/thrombosis (HIT/T) is a common complication of heparin therapy that is caused by antibodies to platelet factor 4 (PF4) complexed with heparin. The immune response is polyclonal and polyspecific, ie, more than one neoepitope on PF4 is recognized by HIT/T antibodies. One such epitope has been previously identified; it involves the domain between the third and fourth cysteine residues in PF4 (site 1). However, the binding sites for other HIT/T antibodies remain to be defined. To explore this issue, the binding site of KKO, an HIT/T-like murine monoclonal antibody, was defined. KKO shares a binding site with many HIT/T antibodies on PF4/heparin, but does not bind to site 1 or recognize mouse PF4/heparin. Therefore, the binding of KKO to a series of mouse/human PF4 chimeras complexed with heparin was examined. KKO recognizes a site that requires both the N terminus of PF4 and Pro34, which immediately precedes the third cysteine. Both regions lie on the surface of the PF4 tetramer in sufficient proximity (within 0.74 nm) to form a contiguous antigenic determinant. The 10 of 14 HIT/T sera that require the N terminus of PF4 for antigen recognition also require Pro34 to bind. This epitope, termed site 2, lies adjacent to site 1 in the crystal structure of the PF4 tetramer. Yet sites 1 and 2 can be recognized by distinct populations of antibodies. These studies further help to define a portion of the PF4 tetramer to which self-reactive antibodies develop in patients exposed to heparin.

Role of the potassium/lysine cationic center in catalysis and functional asymmetry in membrane-bound pyrophosphatases

2018 ◽  
Vol 475 (6) ◽  
pp. 1141-1158 ◽  
Author(s):  
Erika Artukka ◽  
Heidi H. Luoto ◽  
Alexander A. Baykov ◽  
Reijo Lahti ◽  
Anssi M. Malinen
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Systematic Analysis ◽  
Change Mechanism ◽  
Excess Substrate ◽  
Na Ions ◽  
Membrane Bound ◽  
A Site ◽  
Pyrophosphate Hydrolysis

Membrane-bound pyrophosphatases (mPPases), which couple pyrophosphate hydrolysis to transmembrane transport of H+ and/or Na+ ions, are divided into K+,Na+-independent, Na+-regulated, and K+-dependent families. The first two families include H+-transporting mPPases (H+-PPases), whereas the last family comprises one Na+-transporting, two Na+- and H+-transporting subfamilies (Na+-PPases and Na+,H+-PPases, respectively), and three H+-transporting subfamilies. Earlier studies of the few available model mPPases suggested that K+ binds to a site located adjacent to the pyrophosphate-binding site, but is substituted by the ε-amino group of an evolutionarily acquired lysine residue in the K+-independent mPPases. Here, we performed a systematic analysis of the K+/Lys cationic center across all mPPase subfamilies. An Ala → Lys replacement in K+-dependent mPPases abolished the K+ dependence of hydrolysis and transport activities and decreased these activities close to the level (4–7%) observed for wild-type enzymes in the absence of monovalent cations. In contrast, a Lys → Ala replacement in K+,Na+-independent mPPases conferred partial K+ dependence on the enzyme by unmasking an otherwise conserved K+-binding site. Na+ could partially replace K+ as an activator of K+-dependent mPPases and the Lys → Ala variants of K+,Na+-independent mPPases. Finally, we found that all mPPases were inhibited by excess substrate, suggesting strong negative co-operativity of active site functioning in these homodimeric enzymes; moreover, the K+/Lys center was identified as part of the mechanism underlying this effect. These findings suggest that the mPPase homodimer possesses an asymmetry of active site performance that may be an ancient prototype of the rotational binding-change mechanism of F-type ATPases.

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