scholarly journals Water structure and dynamics in the hydration layer of a type III anti-freeze protein

2018 ◽  
Vol 20 (10) ◽  
pp. 6996-7006 ◽  
Author(s):  
Z. Faidon Brotzakis ◽  
Ilja K. Voets ◽  
Huib J. Bakker ◽  
Peter G. Bolhuis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Site ◽  
Water Structure ◽  
Hydration Water ◽  
Tetrahedral Structure ◽  
Structure And Dynamics ◽  
Ice Binding ◽  
Left And Right ◽  
Middle Panel

The tetrahedral structure of hydration water (S) and its reorientation decay time (τ) correlates negatively for selected amino-acids in the vicinity of the ice binding site (left and right panels) of the antifreeze protein, but positively for the ice binding site central amino-acid (middle panel).

scholarly journals Mechanism and site of action of big dynorphin on ASIC1a

2019 ◽  
Author(s):  
C.B. Borg ◽  
N. Braun ◽  
S.A. Heusser ◽  
Y. Bay ◽  
D. Weis ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ischemic Stroke ◽  
Amino Acid ◽  
Binding Site ◽  
Neuronal Death ◽  
Electrostatic Interactions ◽  
Rational Design ◽  
Site Of Action ◽  
Canonical Amino Acid

AbstractAcid-sensing ion channels (ASICs) are proton-gated cation channels that contribute to neurotransmission, as well as initiation of pain and neuronal death following ischemic stroke. As such, there is a great interest in understanding the in vivo regulation of ASICs, especially by endogenous neuropeptides that potently modulate ASICs. The most potent endogenous ASIC modulator known to date is the opioid neuropeptide big dynorphin (BigDyn). BigDyn is upregulated in chronic pain and increases ASIC-mediated neuronal death during acidosis. Understanding the mechanism and site of action of BigDyn on ASICs could thus enable the rational design of compounds potentially useful in the treatment of pain and ischemic stroke. To this end, we employ a combination of electrophysiology, voltage-clamp fluorometry, synthetic BigDyn analogs and non-canonical amino acid-mediated photocrosslinking. We demonstrate that BigDyn binding results in an ASIC1a closed resting conformation that is distinct from open and desensitized states induced by protons. Using alanine-substituted BigDyn analogs, we find that the BigDyn modulation of ASIC1a is mediated through electrostatic interactions of basic amino acids in the BigDyn N-terminus. Furthermore, neutralizing acidic amino acids in the ASIC1a extracellular domain reduces BigDyn effects, suggesting a binding site at the acidic pocket. This is confirmed by photocrosslinking using the non-canonical amino acid azido-phenylalanine. Overall, our data define the mechanism of how BigDyn modulates ASIC1a, identify the acidic pocket as the binding site for BigDyn and thus highlight this cavity as an important site for the development of ASIC-targeting therapeutics.Significance StatementNeuropeptides such as big dynorphin (BigDyn) play important roles in the slow modulation of fast neurotransmission, which is mediated by membrane-embedded receptors. In fact, BigDyn is the most potent known endogenous modulator of one such receptor, the acid-sensing ion channel (ASIC), but the mode of action remains unknown. In this work, we employ a broad array of technologies to unravel the details of where big dynorphin binds to ASIC and how it modulates its activity. As both BigDyn and ASIC are implicated in pain pathways, this work might pave the way towards future analgesics.

scholarly journals Novel alanine serine cysteine transporter 2 (ASCT2) inhibitors based on sulfonamide and sulfonic acid ester scaffolds

2019 ◽  
Vol 151 (3) ◽  
pp. 357-368 ◽  
Author(s):  
Elias Ndaru ◽  
Rachel-Ann A. Garibsingh ◽  
YueYue Shi ◽  
Evan Wallace ◽  
Paul Zakrepine ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Site ◽  
Acid Ester ◽  
Sulfonic Acid ◽  
Chemical Space ◽  
Glutamate Transporter ◽  
Homology Model ◽  
Side Chain ◽  
Large Variability

The neutral amino acid transporter alanine serine cysteine transporter 2 (ASCT2) belongs to the solute carrier 1 (SLC1) family of transport proteins and transports neutral amino acids, such as alanine and glutamine, into the cell in exchange with intracellular amino acids. This amino acid transport is sodium dependent, but not driven by the transmembrane Na+ concentration gradient. Glutamine transport by ASCT2 is proposed to be important for glutamine homoeostasis in rapidly growing cancer cells to fulfill the energy and nitrogen demands of these cells. Thus, ASCT2 is thought to be a potential anticancer drug target. However, the pharmacology of the amino acid binding site is not well established. Here, we report on the synthesis and characterization of a novel class of ASCT2 inhibitors based on an amino acid scaffold with a sulfonamide/sulfonic acid ester linker to a hydrophobic group. The compounds were designed based on an improved ASCT2 homology model using the human glutamate transporter hEAAT1 crystal structure as a modeling template. The compounds were shown to inhibit with a competitive mechanism and a potency that scales with the hydrophobicity of the side chain. The most potent compound binds with an apparent affinity, Ki, of 8 ± 4 µM and can block the alanine response with a Ki of 40 ± 23 µM at 200 µM alanine concentration. Computational analysis predicts inhibitor interactions with the binding site through molecular docking. In conclusion, the sulfonamide/sulfonic acid ester scaffold provides facile synthetic access to ASCT2 inhibitors with a potentially large variability in chemical space of the hydrophobic side chain. These inhibitors will be useful chemical tools to further characterize the role of ASCT2 in disease as well as improve our understanding of inhibition mechanisms of this transporter.

scholarly journals Identification of a mitochondrial-binding site on the N-terminal end of hexokinase II

2015 ◽  
Vol 35 (3) ◽  
Author(s):  
Nadezda Bryan ◽  
Kevin P. Raisch
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Site ◽  
Hexokinase Ii

The first ten amino acids of hexokinase II are import for binding to the MOM. Mutating the fifth amino acid abolished mitochondrial binding. Determining the binding site is important for identifying novel compounds that can disrupt HKII binding to VDAC.

scholarly journals The phtE Locus in the Phaseolotoxin Gene Cluster Has ORFs with Homologies to Genes Encoding Amino Acid Transferases, the AraC Family of Transcriptional Factors, and Fatty Acid Desaturases

1997 ◽  
Vol 10 (8) ◽  
pp. 947-960 ◽  
Author(s):  
Yuan Xin Zhang ◽  
Suresh S. Patil
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Amino Acid ◽  
Binding Site ◽  
Pseudomonas Syringae ◽  
Pyridoxal Phosphate ◽  
Fatty Acid Desaturase ◽  
Transcriptional Factors ◽  
Copper Binding ◽  
Arac Family

A cluster of genes involved in the production of phaseolotoxin, a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, contains eight (phtA through phtH) complementation groups (Y. X. Zhang, K. B. Rowley, and S. S. Patil, J. Bacteriol., 175:6451–6458, 1993). In this study, sequencing of the region encompassing the phtE locus revealed six putative open reading frames (ORFs), each preceded by a putative ribosomal binding site, and all oriented in the same direction. Reverse transcription-polymerase chain reaction suggested that the phtE locus is transcribed as one large (6.4 kb) transcript, indicating that the ORFs constitute an operon. Primer extension analysis showed that the transcript begins at a T, located 31 bp upstream of the ATG codon of ORF1. Comparison of the sequences of the putative ORFs with the sequences of known genes revealed that ORF3, encoding a protein containing 395 amino acids, has 55% similarity to the acetylornithine aminotransferase gene from Escherichia coli, and the ornithine aminotransferase genes from other organisms. A lysine residue that is a binding site for pyridoxal phosphate and an arginine residue that is a binding site for the α-carboxylate group of the substrate are conserved in ORF3. These data suggest that ORF3 encodes a protein involved in the biosynthesis of ornithine, a constituent of phaseolotoxin. ORF5, encoding a peptide of 378 amino acid residues, possesses a helix-turn-helix motif at the C-terminal end that is characteristic of the AraC family of transcriptional factors, and there is a possible leucine zipper at the N-terminal end of this peptide. ORF6, encoding a protein of 327 amino acids, has about 40% similarity with the fatty acid desaturase gene, desA, of Synechocystis Pcc6803 and considerable similarity with fatty acid desaturase genes from other organisms. ORF6 and desA show very similar hydropathy profiles and both contain a copper binding signature. Computer searches did not discover significant homologies in the data base for the other ORFs, but hydropathy analysis showed that all of them contain one to several hydrophobic domains, suggesting that the gene products of these ORFs may be membrane associated.

scholarly journals Investigating the Specific Interactions Between Microtubule Stabilising Agents and β-Tubulin

2021 ◽  
Author(s):  
◽  
Benjamin Jones
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Cell Proliferation ◽  
Amino Acid ◽  
Binding Site ◽  
Drug Class ◽  
Efflux Pumps ◽  
Peloruside A ◽  
Mutant Cells ◽  
Β Tubulin

<p>Microtubule stabilising agents are a class of cytotoxic compounds that cause mitotic arrest through inhibition of microtubule function. They specifically target β-tubulin subunits promoting tubulin polymerisation, which eventually leads to cell death. Members of this drug class include the cancer chemotherapeutics paclitaxel and ixabepilone. However, like many cytotoxic agents, tumour cells often develop multi-drug resistance phenotypes limiting the effectiveness of such compounds. This results from the expulsion of these drugs from cells by efflux pumps, as well as mutation of their binding site. Much effort has been focused on improving the utility of this important drug class in the ongoing fight against cancer.  The microtubule stabilising agents peloruside A and laulimalide originate from marine sponge species native to the South Pacific. They have similar pharmacological profiles to paclitaxel and ixabepilone, however with several unique properties. They are poor substrates for efflux pumps and target a different region on β-tubulin subunits, giving them the potential for treatment of resistant tumours. This represents a novel mechanism of action that may be exploited for drug development, and further characterisation of the binding site is warranted.  The aim of this study is to investigate the contribution of two amino acids of human βItubulin to the interactions with peloruside A and laulimalide. Specifically, glu127 and lys124 have been predicted by computational modelling and analogue studies to form hydrogen bonds and other associations with the two compounds. These amino acids are located on β-tubulin subunits adjacent to the main binding pocket of peloruside A and laulimalide, and represent a potential inter-protofilament interaction that does not occur with other microtubule stabilising agents. This binding mechanism has not yet been shown by crystallography and is hence based solely on in silico work, requiring biological validation.  HEK293 cells were transfected with βI-tubulin with these amino acids mutated to alanines to prevent hydrogen bond formation. Cell proliferation assays, flow cytometry, and immunoblotting were used to study the effect loss of the inter-protofilament interaction has on the bioactivity of peloruside A and laulimalide. These mutations did not significantly alter the concentration-response of cells to either drug in the cell proliferation assay. However, accumulation of cells in the G2/M phase of the cell cycle and the proportion of transfected cells showing signs of mitotic arrest significantly decreased for E127A mutant cells compared to wild type βI-tubulin transfected control cells treated with peloruside A. Furthermore, a similar reduction in cell cycle block was also seen in E127A mutant cells treated with the negative control ixabepilone, which binds to a different site on β-tubulin.  No evidence seen in this study suggests that either amino acid plays a major role in peloruside A or laulimalide target binding. However, the amino acid E127 is important for inter-protofilament associations independent of drug treatment, as its mutation appeared to reduce global stability of microtubule structures. This information requires further validation, it may be useful in the design of future analogue syntheses as development of these promising drug candidates continues.</p>

scholarly journals miR-1322 Binding Sites in Paralogous and Orthologous Genes

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Raigul Niyazova ◽  
Olga Berillo ◽  
Shara Atambayeva ◽  
Anna Pyrkova ◽  
Aigul Alybayeva ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcription Factors ◽  
Amino Acid ◽  
Binding Site ◽  
Binding Sites ◽  
Target Genes ◽  
Amino Acid Sequences ◽  
Orthologous Genes ◽  
Start Position ◽  
Human Genes

We searched for 2,563 microRNA (miRNA) binding sites in 17,494 mRNA sequences of human genes. miR-1322 has more than 2,000 binding sites in 1,058 genes withΔG/ΔGmratio of 85% and more. miR-1322 has 1,889 binding sites in CDSs, 215 binding sites in 5′ UTRs, and 160 binding sites in 3′ UTRs. From two to 28 binding sites have arranged localization with the start position through three nucleotides of each following binding site. The nucleotide sequences of these sites in CDSs encode oligopeptides with the same and/or different amino acid sequences. We found that 33% of the target genes encoded transcription factors. miR-1322 has arranged binding sites in the CDSs of orthologousMAMLD1,MAML2, andMAML3genes. These sites encode a polyglutamine oligopeptide ranging from six to 47 amino acids in length. The properties of miR-1322 binding sites in orthologous and paralogous target genes are discussed.

scholarly journals Significance of conservative asparagine residues in the thermal hysteresis activity of carrot antifreeze protein

2004 ◽  
Vol 377 (3) ◽  
pp. 589-595 ◽  
Author(s):  
Dang-Quan ZHANG ◽  
Bing LIU ◽  
Dong-Ru FENG ◽  
Yan-Ming HE ◽  
Shu-Qi WANG ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Tandem Repeat ◽  
Thermal Hysteresis ◽  
Three Dimensional ◽  
Antifreeze Protein ◽  
Three Dimensional Model ◽  
Prism Plane ◽  
Thermal Hysteresis Activity ◽  
Ice Binding

The ~24-amino-acid leucine-rich tandem repeat motif (PXXXXXLXXLXXLXLSXNXLXGXI) of carrot antifreeze protein comprises most of the processed protein and should contribute at least partly to the ice-binding site. Structural predictions using publicly available online sources indicated that the theoretical three-dimensional model of this plant protein includes a 10-loop β-helix containing the ~24-amino-acid tandem repeat. This theoretical model indicated that conservative asparagine residues create putative ice-binding sites with surface complementarity to the 1010 prism plane of ice. We used site-specific mutagenesis to test the importance of these residues, and observed a distinct loss of thermal hysteresis activity when conservative asparagines were replaced with valine or glutamine, whereas a large increase in thermal hysteresis was observed when phenylalanine or threonine residues were replaced with asparagine, putatively resulting in the formation of an ice-binding site. These results confirmed that the ice-binding site of carrot antifreeze protein consists of conservative asparagine residues in each β-loop. We also found that its thermal hysteresis activity is directly correlated with the length of its asparagine-rich binding site, and hence with the size of its ice-binding face.

Structure-based characterization and antifreeze properties of a hyperactive ice-binding protein from the Antarctic bacteriumFlavobacterium frigorisPS1

2014 ◽  
Vol 70 (4) ◽  
pp. 1061-1073 ◽  
Author(s):  
Hackwon Do ◽  
Soon-Jong Kim ◽  
Hak Jun Kim ◽  
Jun Hyuck Lee
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Disulfide Bond ◽  
Binding Protein ◽  
Size Exclusion ◽  
Sequence Alignments ◽  
Ice Binding ◽  
Ice Binding Protein ◽  
Binding Residues ◽  
The Antarctic

Ice-binding proteins (IBPs) inhibit ice growth through direct interaction with ice crystals to permit the survival of polar organisms in extremely cold environments. FfIBP is an ice-binding protein encoded by the Antarctic bacteriumFlavobacterium frigorisPS1. The X-ray crystal structure of FfIBP was determined to 2.1 Å resolution to gain insight into its ice-binding mechanism. The refined structure of FfIBP shows an intramolecular disulfide bond, and analytical ultracentrifugation and analytical size-exclusion chromatography show that it behaves as a monomer in solution. Sequence alignments and structural comparisons of IBPs allowed two groups of IBPs to be defined, depending on sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. Although FfIBP closely resemblesLeucosporidium(recently re-classified asGlaciozyma) IBP (LeIBP) in its amino-acid sequence, the thermal hysteresis (TH) activity of FfIBP appears to be tenfold higher than that of LeIBP. A comparison of the FfIBP and LeIBP structures reveals that FfIBP has different ice-binding residues as well as a greater surface area in the ice-binding site. Notably, the ice-binding site of FfIBP is composed of a T-A/G-X-T/N motif, which is similar to the ice-binding residues of hyperactive antifreeze proteins. Thus, it is proposed that the difference in TH activity between FfIBP and LeIBP may arise from the amino-acid composition of the ice-binding site, which correlates with differences in affinity and surface complementarity to the ice crystal. In conclusion, this study provides a molecular basis for understanding the antifreeze mechanism of FfIBP and provides new insights into the reasons for the higher TH activity of FfIBP compared with LeIBP.

scholarly journals The Venus Fly Trap Domain of the Extracellular Ca2+-sensing Receptor Is Required for l-Amino Acid Sensing

2004 ◽  
Vol 279 (50) ◽  
pp. 51739-51744 ◽  
Author(s):  
Hee-Chang Mun ◽  
Alison H. Franks ◽  
Emma L. Culverston ◽  
Karen Krapcho ◽  
Edward F. Nemeth ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Site ◽  
Hek293 Cells ◽  
Transmembrane Region ◽  
Rich Region ◽  
Metabotropic Glutamate ◽  
C Terminus ◽  
Amino Acid Binding ◽  
Amino Acid Sensing

We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated byl-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000)Proc. Natl. Acad. Sci. U. S. A.97, 4814–4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation byl-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1–599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond tol-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retainedl-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary forl-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site ofl-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site forl-glutamate is conserved as anl-amino acid binding site in its homolog, the CaR.

scholarly journals Structure of RizA, anL-amino-acid ligase fromBacillus subtilis

2015 ◽  
Vol 71 (9) ◽  
pp. 1125-1130 ◽  
Author(s):  
Wataru Kagawa ◽  
Toshinobu Arai ◽  
Shun Ishikura ◽  
Kuniki Kino ◽  
Hitoshi Kurumizaka
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Binding Site ◽  
Free Form ◽  
Biochemical Studies ◽  
Amino Acid Binding

RizA is an L-amino-acid ligase fromBacillus subtilisthat participates in the biosynthesis of rhizocticin, an oligopeptide antibiotic. The substrate-free form of RizA has been crystallized and the structure was solved at 2.8 Å resolution. The amino-acid-binding site appears to be capable of accommodating multiple amino acids, consistent with previous biochemical studies.

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