Fine mapping of the alpha-actinin binding site within cysteine-rich protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Harper BD
PROVIDER: S-EPMC1221251 | biostudies-other | 2000 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The assembly and deposition of amyloid β-protein (Aβ) in brain is a key pathological feature of Alzheimer's disease and related disorders. Factors have been identified that can either promote or inhibit Aβ assembly in brain. We previously reported that myelin basic protein (MBP) is a potent inhibitor of Aβ fibrillar assembly [Hoos, M. D., et al. (2007) J. Biol. Chem. 282, 9952-9961; Hoos, M. D., et al. (2009) Biochemistry 48, 4720-4727]. Moreover, the region on MBP responsible for this activity was localized to the 64 N-terminal amino acids (MBP1-64) [Liao, M. C., et al. (2010) J. Biol. Chem. 285, 35590-35598]. In the study presented here, we sought to further define the site on MBP1-64 involved in this activity. Deletion mapping studies showed that the C-terminal region (residues 54-64) is required for the ability of MBP1-64 to bind Aβ and inhibit fibril assembly. Alanine scanning mutagenesis revealed that amino acids K54, R55, G56, and K59 within MBP1-64 are important for both Aβ binding and inhibition of fibril assembly as assessed by solid phase binding, thioflavin T binding and fluorescence, and transmission electron microscopy studies. Strong spectral shifts are observed by solution nuclear magnetic resonance spectroscopy of specific N-terminal residues (E3, R5, D7, E11, and Q15) of Aβ42 upon the interaction with MBP1-64. Although the C-terminal region of MBP1-64 is required for interactions with Aβ, a synthetic MBP50-64 peptide was itself devoid of activity. These studies identify key residues in MBP and Aβ involved in their interactions and provide structural insight into how MBP regulates Aβ fibrillar assembly.
| S-EPMC3640357 | biostudies-literature
Project description:Using low-speed sedimentation equilibrium we have established that vinculin binds to alpha-actinin with a Kd of 1.3 x 10(-5) M. Electron microscopy of negatively stained preparations of vinculin revealed spherical particles (diameter 11.2 nm; S.D. 1.7 nm, n = 21), whereas alpha-actinin appeared as a rod-shaped particle (length 33 nm; S.D. 3.3 nm, n = 23). Mixtures of the two proteins contained both 'lollipop'- and 'dumbell'-shaped particles which we interpret as either one or two spherical vinculin molecules associated with the ends of the alpha-actinin rod. We have further defined the vinculin-binding site in alpha-actinin using 125I-vinculin and a gel-blot assay in which proteolytic fragments of alpha-actinin and fragments of alpha-actinin expressed in Escherichia coli were resolved by SDS/PAGE and blotted to nitrocellulose. 125I-vinculin bound to polypeptides derived from the spectrin-like repeat region of alpha-actinin, but did not bind to the actin-binding domain. Binding was inhibited by a 100-fold molar excess of unlabelled vinculin. Using a series of glutathione S-transferase fusion proteins we have mapped the vinculin-binding site to a region toward the C-terminal end of the molecule (alpha-actinin residues 713-749). 125I-vinculin also bound to fusion proteins containing this sequence which had been immobilized on glutathione-agarose beads. The vinculin-binding site is localized in a highly conserved region of the molecule close to the first of two EF-hand calcium-binding motifs.
| S-EPMC1137166 | biostudies-other
Project description:Protein kinase C (PKC) is an important signal transduction protein whose cysteine-rich regulatory domain C1 has been proposed to interact with general anesthetics in both of its diacylglycerol/phorbol ester-binding subdomains, the tandem repeats C1A and C1B. Previously, we identified an allosteric binding site on one of the two cysteine-rich domains, PKCdelta C1B. To test the hypothesis that there is an additional anesthetic site on the other cysteine-rich subdomain, C1A, we subcloned, expressed in Escherichia coli, purified, and characterized mouse PKCdelta C1A. Octanol and butanol both quenched the intrinsic fluorescence of PKCdelta C1A in a saturable manner, suggesting the presence of a binding site. To locate this site, PKCdelta C1A was photolabeled with three diazirine-containing alkanols, 3-azioctanol, 7-azioctanol, and 3-azibutanol. Mass spectrometry revealed that at low concentrations all three photoincorporated into PKCdelta C1A with a stoichiometry of 1:1 in the labeled fraction, but higher stoichiometries occurred at higher concentrations, particularly with azibutanol. Photocomplexes of PKCdelta C1A with azioctanols were separated from the unlabeled protein by HPLC, reduced, alkylated, digested with trypsin, and sequenced by mass spectrometry. All the azioctanols photolabeled PKCdelta C1A at residue Tyr-29, corresponding to Tyr-187 of the full-length PKCdelta, and at a neighboring residue, Lys-40, suggesting there is an alcohol site in this vicinity. In addition, Glu-2 was photolabeled more efficiently by 3-azibutanol than by the azioctanols, suggesting the existence of a second, smaller site.
| S-EPMC2242605 | biostudies-literature
Project description:ATP is the native agonist for cell-surface ligand-gated P2X receptor (P2XR) cation channels. The seven mammalian subunits (P2X1-7) form homo- and heterotrimeric P2XRs having significant physiological and pathophysiological roles. Pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) is an effective antagonist at most mammalian P2XRs. Lys-249 in the extracellular domain of P2XR has previously been shown to contribute to PPADS action. To map this antagonist site, we generated human P2X1R cysteine substitutions within a circle centered at Lys-249 (with a radius of 13 Å equal to the length of PPADS). We hypothesized that cysteine substitutions of residues involved in PPADS binding would (i) reduce cysteine accessibility (measured by MTSEA-biotinylation), (ii) exhibit altered PPADS affinity, and (iii) quench the fluorescence of cysteine residues modified with MTS-TAMRA. Of the 26 residues tested, these criteria were met by only four (Lys-70, Asp-170, Lys-190, and Lys-249), defining the antagonist site, validating molecular docking results, and thereby providing the first experimentally supported model of PPADS binding. This binding site overlapped with the ATP-binding site, indicating that PPADS sterically blocks agonist access. Moreover, PPADS induced a conformational change at the cysteine-rich head (CRH) region adjacent to the orthosteric ATP-binding pocket. The importance of this movement was confirmed by demonstrating that substitution introducing positive charge present in the CRH of the hP2X1R causes PPADS sensitivity at the normally insensitive rat P2X4R. This study provides a template for developing P2XR subtype selectivity based on the differences among the mammalian subunits around the orthosteric P2XR-binding site and the CRH.
| S-EPMC6102130 | biostudies-literature
Project description:Elucidating the principles governing anesthetic-protein interactions requires structural determinations at high resolutions not yet achieved with ion channels. Protein kinase C (PKC) activity is modulated by general anesthetics. We solved the structure of the phorbol-binding domain (C1B) of PKC? complexed with an ether (methoxymethylcycloprane) and with an alcohol (cyclopropylmethanol) at 1.36-Å resolution. The cyclopropane rings of both agents displace a single water molecule in a surface pocket adjacent to the phorbol-binding site, making van der Waals contacts with the backbone and/or side chains of residues Asn-237 to Ser-240. Surprisingly, two water molecules anchored in a hydrogen-bonded chain between Thr-242 and Lys-260 impart elasticity to one side of the binding pocket. The cyclopropane ring takes part in ?-acceptor hydrogen bonds with the amide of Met-239. There is a crucial hydrogen bond between the oxygen atoms of the anesthetics and the hydroxyl of Tyr-236. A Tyr-236-Phe mutation results in loss of binding. Thus, both van der Waals interactions and hydrogen-bonding are essential for binding to occur. Ethanol failed to bind because it is too short to benefit from both interactions. Cyclopropylmethanol inhibited phorbol-ester-induced PKC? activity, but failed to do so in PKC? containing the Tyr-236-Phe mutation.
| S-EPMC3514512 | biostudies-literature
Project description:To better understand the structure and function of Z lines, we used sarcomeric isoforms of alpha-actinin and gamma-filamin to screen a human skeletal muscle cDNA library for interacting proteins by using the yeast two-hybrid system. Here we describe myozenin (MYOZ), an alpha-actinin- and gamma-filamin-binding Z line protein expressed predominantly in skeletal muscle. Myozenin is predicted to be a 32-kDa, globular protein with a central glycine-rich domain flanked by alpha-helical regions with no strong homologies to any known genes. The MYOZ gene has six exons and maps to human chromosome 10q22.1-q22.2. Northern blot analysis demonstrated that this transcript is expressed primarily in skeletal muscle with significantly lower levels of expression in several other tissues. Antimyozenin antisera stain skeletal muscle in a sarcomeric pattern indistinguishable from that seen by using antibodies for alpha-actinin, and immunogold electron microscopy confirms localization specifically to Z lines. Thus, myozenin is a skeletal muscle Z line protein that may be a good candidate gene for limb-girdle muscular dystrophy or other neuromuscular disorders.
| S-EPMC29302 | biostudies-literature
Project description:Cell adhesion is tightly regulated by specific molecular interactions and detachment from the extracellular matrix modifies proliferation and survival. HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a protein-lipid complex with tumoricidal activity that also triggers tumor cell detachment in vitro and in vivo, suggesting that molecular interactions defining detachment are perturbed in cancer cells. To identify such interactions, cell membrane extracts were used in Far-western blots and HAMLET was shown to bind α-actinins; major F-actin cross-linking proteins and focal adhesion constituents. Synthetic peptide mapping revealed that HAMLET binds to the N-terminal actin-binding domain as well as the integrin-binding domain of α-actinin-4. By co-immunoprecipitation of extracts from HAMLET-treated cancer cells, an interaction with α-actinin-1 and -4 was observed. Inhibition of α-actinin-1 and α-actinin-4 expression by siRNA transfection increased detachment, while α-actinin-4-GFP over-expression significantly delayed rounding up and detachment of tumor cells in response to HAMLET. In response to HAMLET, adherent tumor cells rounded up and detached, suggesting a loss of the actin cytoskeletal organization. These changes were accompanied by a reduction in β1 integrin staining and a decrease in FAK and ERK1/2 phosphorylation, consistent with a disruption of integrin-dependent cell adhesion signaling. Detachment per se did not increase cell death during the 22 hour experimental period, regardless of α-actinin-4 and α-actinin-1 expression levels but adherent cells with low α-actinin levels showed increased death in response to HAMLET. The results suggest that the interaction between HAMLET and α-actinins promotes tumor cell detachment. As α-actinins also associate with signaling molecules, cytoplasmic domains of transmembrane receptors and ion channels, additional α-actinin-dependent mechanisms are discussed.
| S-EPMC3050841 | biostudies-literature
Project description:Many microorganisms in Antarctica survive in the cold environment there by producing ice-binding proteins (IBPs) to control the growth of ice around them. An IBP from the Antarctic freshwater microalga, Chloromonas sp., was identified and characterized. The length of the Chloromonas sp. IBP (ChloroIBP) gene was 3.2 kb with 12 exons, and the molecular weight of the protein deduced from the ChloroIBP cDNA was 34.0 kDa. Expression of the ChloroIBP gene was up- and down-regulated by freezing and warming conditions, respectively. Western blot analysis revealed that native ChloroIBP was secreted into the culture medium. This protein has fifteen cysteines and is extensively disulfide bonded as shown by in-gel mobility shifts between oxidizing and reducing conditions. The open-reading frame of ChloroIBP was cloned and over-expressed in Escherichia coli to investigate the IBP's biochemical characteristics. Recombinant ChloroIBP produced as a fusion protein with thioredoxin was purified by affinity chromatography and formed single ice crystals of a dendritic shape with a thermal hysteresis activity of 0.4±0.02°C at a concentration of 5 mg/ml. In silico structural modeling indicated that the three-dimensional structure of ChloroIBP was that of a right-handed ?-helix. Site-directed mutagenesis of ChloroIBP showed that a conserved region of six parallel T-X-T motifs on the ?-2 face was the ice-binding region, as predicted from the model. In addition to disulfide bonding, hydrophobic interactions between inward-pointing residues on the ?-1 and ?-2 faces, in the region of ice-binding motifs, were crucial to maintaining the structural conformation of ice-binding site and the ice-binding activity of ChloroIBP.
| S-EPMC4838330 | biostudies-literature
Project description:BackgroundCross-linking and immunoprecipitation followed by next-generation sequencing (CLIP-seq) is the state-of-the-art technique used to experimentally determine transcriptome-wide binding sites of RNA-binding proteins (RBPs). However, it relies on gene expression, which can be highly variable between conditions and thus cannot provide a complete picture of the RBP binding landscape. This creates a demand for computational methods to predict missing binding sites. Although there exist various methods using traditional machine learning and lately also deep learning, we encountered several problems: many of these are not well documented or maintained, making them difficult to install and use, or are not even available. In addition, there can be efficiency issues, as well as little flexibility regarding options or supported features.ResultsHere, we present RNAProt, an efficient and feature-rich computational RBP binding site prediction framework based on recurrent neural networks. We compare RNAProt with 1 traditional machine learning approach and 2 deep-learning methods, demonstrating its state-of-the-art predictive performance and better run time efficiency. We further show that its implemented visualizations capture known binding preferences and thus can help to understand what is learned. Since RNAProt supports various additional features (including user-defined features, which no other tool offers), we also present their influence on benchmark set performance. Finally, we show the benefits of incorporating additional features, specifically structure information, when learning the binding sites of an hairpin loop binding RBP.ConclusionsRNAProt provides a complete framework for RBP binding site predictions, from data set generation over model training to the evaluation of binding preferences and prediction. It offers state-of-the-art predictive performance, as well as superior run time efficiency, while at the same time supporting more features and input types than any other tool available so far. RNAProt is easy to install and use, comes with comprehensive documentation, and is accompanied by informative statistics and visualizations. All this makes RNAProt a valuable tool to apply in future RBP binding site research.
| S-EPMC8372218 | biostudies-literature
Project description:The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker ?-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease.
| S-EPMC4450414 | biostudies-literature