Identification of a novel microtubule binding and assembly domain in the developmentally regulated inter-repeat region of tau

Tau is a developmentally regulated microtubule-associated protein that influences microtubule behavior by directly associating with tubulin. The carboxyl terminus of tau contains multiple 18-amino acid repeats that bind microtubules and are separated by 13-14-amino acid inter-repeat (IR) regions previously thought to function as "linkers." Here, we have performed a high resolution deletion analysis of tau and identified the IR region located between repeats 1 and 2 (the R1-R2 IR) as a unique microtubule binding site with more than twice the binding affinity of any individual repeat. Truncation analyses and site-directed mutagenesis reveal that the binding activity of this site is derived primarily from lys265 and lys272, with a lesser contribution from lys271. These results predict strong, discrete electrostatic interactions between the R1-R2 IR and tubulin, in contrast to the distributed array of weak interactions thought to underlie the association between 18-amino acid repeats and microtubules (Butner, K. A., and M. W. Kirschner. J. Cell Biol. 115:717-730). Moreover, competition assays suggest that the R1-R2 IR associates with microtubules at tubulin site(s) distinct from those bound by the repeats. Finally, a synthetic peptide corresponding to just 10 amino acids of the R1-R2 IR is sufficient to promote tubulin polymerization in a sequence-dependent manner. Since the R1-R2 IR is specifically expressed in adult tau, its action may underlie some of the developmental transitions observed in neuronal microtubule organization. We suggest that the R1-R2 IR may establish an adult-specific, high affinity anchor that tethers the otherwise mobile tau molecule to the tubulin lattice, thereby increasing microtubule stability. Moreover, the absence of R1-R2 IR expression during early development may allow for the cytoskeletal plasticity required of immature neurons.

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Identification of the Binding Domain of Streptococcus oralis Glyceraldehyde-3-Phosphate Dehydrogenase for Porphyromonas gingivalis Major Fimbriae

Infection and Immunity ◽

10.1128/iai.00439-09 ◽

2009 ◽

Vol 77 (11) ◽

pp. 5130-5138 ◽

Cited By ~ 24

Author(s):

Hideki Nagata ◽

Mio Iwasaki ◽

Kazuhiko Maeda ◽

Masae Kuboniwa ◽

Ei Hashino ◽

...

Keyword(s):

Amino Acid ◽

Porphyromonas Gingivalis ◽

Biofilm Formation ◽

Binding Activity ◽

Binding Domain ◽

Laser Scanning Microscopy ◽

Dependent Manner ◽

Oral Streptococci ◽

Amino Acid Residues ◽

Streptococcus Oralis

ABSTRACT Porphyromonas gingivalis forms communities with antecedent oral biofilm constituent streptococci. P. gingivalis major fimbriae bind to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) present on the streptococcal surface, and this interaction plays an important role in P. gingivalis colonization. This study identified the binding domain of Streptococcus oralis GAPDH for P. gingivalis fimbriae. S. oralis recombinant GAPDH (rGAPDH) was digested with lysyl endopeptidase. Cleaved fragments of rGAPDH were applied to a reverse-phase high-pressure liquid chromatograph equipped with a C18 column. Each peak was collected; the binding activity toward P. gingivalis recombinant fimbrillin (rFimA) was analyzed with a biomolecular interaction analysis system. The fragment displaying the strongest binding activity was further digested with various proteinases, after which the binding activity of each fragment was measured. The amino acid sequence of each fragment was determined by direct sequencing, mass spectrometric analysis, and amino acid analysis. Amino acid residues 166 to 183 of S. oralis GAPDH exhibited the strongest binding activity toward rFimA; confocal laser scanning microscopy revealed that the synthetic peptide corresponding to amino acid residues 166 to 183 of S. oralis GAPDH (pep166-183, DNFGVVEGLMTTIHAYTG) inhibits S. oralis-P. gingivalis biofilm formation in a dose-dependent manner. Moreover, pep166-183 inhibited interbacterial biofilm formation by several oral streptococci and P. gingivalis strains with different types of FimA. These results indicate that the binding domain of S. oralis GAPDH for P. gingivalis fimbriae exists within the region encompassing amino acid residues 166 to 183 of GAPDH and that pep166-183 may be a potent inhibitor of P. gingivalis colonization in the oral cavity.

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Fibronectin Binding to the Salmonella enterica Serotype Typhimurium ShdA Autotransporter Protein Is Inhibited by a Monoclonal Antibody Recognizing the A3 Repeat

Journal of Bacteriology ◽

10.1128/jb.186.15.4931-4939.2004 ◽

2004 ◽

Vol 186 (15) ◽

pp. 4931-4939 ◽

Cited By ~ 27

Author(s):

Robert A. Kingsley ◽

Daad Abi Ghanem ◽

Nahum Puebla-Osorio ◽

A. Marijke Keestra ◽

Luc Berghman ◽

...

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Binding Activity ◽

Immune Serum ◽

Amino Acid Residues ◽

Repeat Region ◽

Passenger Domain ◽

Amino Acid Repeats ◽

Primary Amino ◽

Fibronectin Binding

ABSTRACT ShdA is a large outer membrane protein of the autotransporter family whose passenger domain binds the extracellular matrix proteins fibronectin and collagen I, possibly by mimicking the host ligand heparin. The ShdA passenger domain consists of ∼1,500 amino acid residues that can be divided into two regions based on features of the primary amino acid sequence: an N-terminal nonrepeat region followed by a repeat region composed of two types of imperfect direct amino acid repeats, called type A and type B. The repeat region bound bovine fibronectin with an affinity similar to that for the complete ShdA passenger domain, while the nonrepeat region exhibited comparatively low fibronectin-binding activity. A number of fusion proteins containing truncated fragments of the repeat region did not bind bovine fibronectin. However, binding of the passenger domain to fibronectin was inhibited in the presence of immune serum raised to one truncated fragment of the repeat region that contained repeats A2, B8, A3, and B9. Furthermore, a monoclonal antibody that specifically recognized an epitope in a recombinant protein containing the A3 repeat inhibited binding of ShdA to fibronectin.

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Involvement of the N Terminus of Ribosomal Protein L11 in Regulation of the RelA Protein of Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.183.22.6532-6537.2001 ◽

2001 ◽

Vol 183 (22) ◽

pp. 6532-6537 ◽

Cited By ~ 26

Author(s):

Xiaoming Yang ◽

Edward E. Ishiguro

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Ribosomal Protein ◽

Stringent Response ◽

Direct Interaction ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Ribosome Binding ◽

N Terminus ◽

Ribosomal Protein L11

ABSTRACT Amino acid-deprived rplK (previously known asrelC) mutants of Escherichia coli cannot activate (p)ppGpp synthetase I (RelA) and consequently exhibit relaxed phenotypes. The rplK gene encodes ribosomal protein L11, suggesting that L11 is involved in regulating the activity of RelA. To investigate the role of L11 in the stringent response, a derivative ofrplK encoding L11 lacking the N-terminal 36 amino acids (designated ′L11) was constructed. Bacteria overexpressing ′L11 exhibited a relaxed phenotype, and this was associated with an inhibition of RelA-dependent (p)ppGpp synthesis during amino acid deprivation. In contrast, bacteria overexpressing normal L11 exhibited a typical stringent response. The overexpressed ′L11 was incorporated into ribosomes and had no effect on the ribosome-binding activity of RelA. By several methods (yeast two-hybrid, affinity blotting, and copurification), no direct interaction was observed between the C-terminal ribosome-binding domain of RelA and L11. To determine whether the proline-rich helix of L11 was involved in RelA regulation, the Pro-22 residue was replaced with Leu by site-directed mutagenesis. The overexpression of the Leu-22 mutant derivative of L11 resulted in a relaxed phenotype. These results indicate that the proline-rich helix in the N terminus of L11 is involved in regulating the activity of RelA.

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Characterization of the kainate-binding domain of the glutamate receptor GluR-6 subunit

Biochemical Journal ◽

10.1042/bj3301461 ◽

1998 ◽

Vol 330 (3) ◽

pp. 1461-1467 ◽

Cited By ~ 30

Author(s):

Kari KEINÄNEN ◽

Annukka JOUPPILA ◽

Arja KUUSINEN

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Binding Capacity ◽

Binding Activity ◽

Binding Domain ◽

Dependent Manner ◽

Structural Determinants ◽

Concentration Dependent Manner ◽

Binding Characteristics ◽

Linker Peptide

Recombinant fragments of the kainate-selective glutamate recep-tor subunit GluR-6 were expressed in insect cells and analysed for [3H]kainate binding activity in order to characterize the structural determinants responsible for ligand recognition. Deletion of the N-terminal ~ 400 amino-acid-residue segment and the C-terminal ~ 90 residues resulted in a membrane-bound core fragment which displayed pharmacologically native-like [3H]kainate binding properties. Further replacement of the membrane-embedded segments M1-M3 by a hydrophilic linker peptide gave rise to a soluble polypeptide which was accumulated in the culture medium. When bound to chelating Sepharose beads via a C-terminal histidine tag, the soluble fragment showed low-affinity binding of [3H]kainate, which was displaced in a concentration-dependent manner by unlabelled domoic acid, L-glutamate and 6-cyano-7-nitroquinoxaline-2,3-dione. Our results indicate that the kainate-binding site is formed exclusively by the two discontinuous extracellular segments (S1 and S2) which are homologous to bacterial amino-acid-binding proteins. Ligand binding characteristics of soluble S1-S2 chimaeras between the GluR-6 and GluR-D subunits showed that, whereas both S1 and S2 segments contribute to agonist-selectivity, the N-terminal one-third of the GluR-D S2 segment is sufficient to confer α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-binding capacity to the chimaeric ligand-binding domain.

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Identification of the pH sensor and activation by chemical modification of the ClC-2G Cl− channel

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.4.c1113 ◽

1998 ◽

Vol 275 (4) ◽

pp. C1113-C1123 ◽

Cited By ~ 33

Author(s):

Katarina Stroffekova ◽

Elena Y. Kupert ◽

Danuta H. Malinowska ◽

John Cuppoletti

Keyword(s):

Amino Acid ◽

Ph Sensor ◽

Extracellular Ph ◽

Site Directed Mutagenesis ◽

Acid Activation ◽

Dependent Manner ◽

Open Probability ◽

Concentration Dependent Manner ◽

Time Constants ◽

Extracellular Region

Rabbit and human ClC-2G Cl− channels are voltage sensitive and activated by protein kinase A and low extracellular pH. The objective of the present study was to investigate the mechanism involved in acid activation of the ClC-2G Cl− channel and to determine which amino acid residues play a role in this acid activation. Channel open probability ( P o) at ±80 mV holding potentials increased fourfold in a concentration-dependent manner with extracellular H+concentration (that is, extracellular pH, pH trans ), with an apparent acidic dissociation constant of pH 4.95 ± 0.27. 1-Ethyl-3(3-dimethylaminopropyl)carbodiimide-catalyzed amidation of the channel with glycine methyl ester increased P o threefold at pH trans 7.4, at which the channel normally exhibits low P o. With extracellular pH reduction (protonation) or amidation, increased P o was due to a significant increase in open time constants and a significant decrease in closed time constants of the channel gating, and this effect was insensitive to applied voltage. With the use of site-directed mutagenesis, the extracellular region EELE (amino acids 416–419) was identified as the pH sensor and amino acid Glu-419 was found to play the key or predominant role in activation of the ClC-2G Cl− channel by extracellular acid.

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The EF-hand Ca2+-binding Protein p22 Associates with Microtubules in an N-Myristoylation–dependent Manner

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3473 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3473-3488 ◽

Cited By ~ 34

Author(s):

Sandy Timm ◽

Brian Titus ◽

Karen Bernd ◽

Margarida Barroso

Keyword(s):

Binding Protein ◽

Gel Filtration ◽

Membrane Traffic ◽

Binding Activity ◽

Binding Motif ◽

Dependent Manner ◽

Microtubule Cytoskeleton ◽

Microtubule Binding ◽

Ef Hand ◽

Associated Proteins

Proteins containing the EF-hand Ca2+-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca2+-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22–microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation–dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22’s Ca2+-binding activity, suggesting that the p22–microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

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Molecular Genetic Evidence that the Hydrophobic Anchors of Glycoproteins E2 and E1 Interact during Assembly of Alphaviruses

Journal of Virology ◽

10.1128/jvi.76.20.10188-10194.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10188-10194 ◽

Cited By ~ 18

Author(s):

Ellen G. Strauss ◽

Edith M. Lenches ◽

James H. Strauss

Keyword(s):

Amino Acid ◽

Sindbis Virus ◽

Transmembrane Domain ◽

Molecular Genetic ◽

Site Directed Mutagenesis ◽

Cumulative Effects ◽

Ross River Virus ◽

Directed Mutagenesis ◽

Dependent Manner ◽

Molecular Genetic Evidence

ABSTRACT Chimeric alphaviruses in which the 6K and glycoprotein E1 moieties of Sindbis virus are replaced with those of Ross River virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. We have sequenced the entire domain encoding the E2, 6K, and E1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. In three independent passage series, amino acid 380 of E2, which is in the transmembrane domain, was mutated from the original isoleucine to serine in two instances and to valine once. We have now changed this residue to seven others by site-directed mutagenesis and tested the effects of these mutations on the growth of both the chimera [SIN(RRE1)] and of parental Sindbis. These results indicate that the transmembrane domains of glycoproteins E2 and E1 of alphaviruses interact in a sequence-dependent manner and that this interaction is required for efficient budding and assembly of infectious virions.

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Functional interactions between the proline-rich and repeat regions of tau enhance microtubule binding and assembly.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.353 ◽

1997 ◽

Vol 8 (2) ◽

pp. 353-365 ◽

Cited By ~ 176

Author(s):

B L Goode ◽

P E Denis ◽

D Panda ◽

M J Radeke ◽

H P Miller ◽

...

Keyword(s):

Amino Acid ◽

Structural Complexity ◽

Intramolecular Interactions ◽

Site Directed Mutagenesis ◽

Microtubule Assembly ◽

Repeat Region ◽

Rich Region ◽

Microtubule Binding ◽

Amino Terminal ◽

Proline Rich Region

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.

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