Full-length structure of the major autolysin LytA

LytA is responsible for the autolysis of manyStreptococcusspecies, including pathogens such asS. pneumoniae,S. pseudopneumoniaeandS. mitis. However, how this major autolysin achieves full activity remains unknown. Here, the full-length structure of theS. pneumoniaeLytA dimer is reported at 2.1 Å resolution. Each subunit has an N-terminal amidase domain and a C-terminal choline-binding domain consisting of six choline-binding repeats, which form five canonical and one single-layered choline-binding sites. Site-directed mutageneses combined with enzymatic activity assays indicate that dimerization and binding to choline are two independent requirements for the autolytic activity of LytAin vivo. Altogether, it is suggested that dimerization and full occupancy of all choline-binding sites through binding to choline-containing TA chains enable LytA to adopt a fully active conformation which allows the amidase domain to cleave two lactyl-amide bonds located about 103 Å apart on the peptidoglycan.

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Microtubule Actin Cross-Linking Factor (Macf)

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1275 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1275-1286 ◽

Cited By ~ 150

Author(s):

Conrad L. Leung ◽

Dongming Sun ◽

Min Zheng ◽

David R. Knowles ◽

Ronald K.H. Liem

Keyword(s):

Calcium Binding ◽

Coiled Coil ◽

Specific Protein ◽

Binding Domain ◽

Full Length ◽

Actin Binding ◽

Cross Linking ◽

Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00351.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. G328-G339 ◽

Cited By ~ 41

Author(s):

P. Singh ◽

X. Lu ◽

S. Cobb ◽

B. T. Miller ◽

N. Tarasova ◽

...

Keyword(s):

Epithelial Cells ◽

Binding Sites ◽

Intestinal Epithelial Cells ◽

Full Length ◽

Intestinal Epithelial ◽

Growth Effects ◽

High Affinity ◽

Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

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Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.12.6663 ◽

1995 ◽

Vol 15 (12) ◽

pp. 6663-6669 ◽

Cited By ~ 50

Author(s):

L Trieschmann ◽

Y V Postnikov ◽

A Rickers ◽

M Bustin

Keyword(s):

Amino Acids ◽

Structural Features ◽

Binding Domain ◽

Full Length ◽

Modular Structure ◽

Chromosomal Proteins ◽

Nucleosome Core ◽

Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

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Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205

Molecular Endocrinology ◽

10.1210/mend.16.7.0883 ◽

2002 ◽

Vol 16 (7) ◽

pp. 1502-1510 ◽

Cited By ~ 15

Author(s):

Yutaka Maeda ◽

Christophe Rachez ◽

Leo Hawel ◽

Craig V. Byus ◽

Leonard P. Freedman ◽

...

Keyword(s):

Vitamin D ◽

Ligand Binding ◽

Nuclear Receptors ◽

Vitamin D Receptor ◽

Binding Domain ◽

Ligand Binding Domain ◽

Full Length ◽

Dependent Manner ◽

Interacting Protein

Abstract Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF–2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4α (HNF4α) activate transcription. In this study, we show that HNF4α interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4α in vivo, it did not interact well with the HNF4α ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4α and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4α and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4α-mediated transcription and that a specific inhibitor of polyamine biosynthesis, α-difluoromethylornithine (DFMO), decreased the ability of HNF4α to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.

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The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

eLife ◽

10.7554/elife.03648 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 43

Author(s):

Gunther Hollopeter ◽

Jeffrey J Lange ◽

Ying Zhang ◽

Thien N Vu ◽

Mingyu Gu ◽

...

Keyword(s):

Binding Sites ◽

Active Conformation ◽

Amino Acid Motif ◽

Bar Domain ◽

Open Conformation ◽

Endocytic Machinery ◽

Associated Proteins ◽

Compensatory Mutations ◽

Clathrin Adaptor

The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2.

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SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection

PLoS Pathogens ◽

10.1371/journal.ppat.1010175 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010175

Author(s):

Abigael Eva Chaouat ◽

Hagit Achdout ◽

Inbal Kol ◽

Orit Berhani ◽

Gil Roi ◽

...

Keyword(s):

Virus Infection ◽

Enzymatic Activity ◽

Receptor Binding ◽

Viral Entry ◽

Surface Expression ◽

Binding Domain ◽

Host Cells ◽

Receptor Binding Domain ◽

High Virus

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus membrane binds to the angiotensin converting enzyme 2) ACE2 (receptor on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

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Ability of scalloped deletion constructs to rescue sd mutant wing phenotypes in Drosophila melanogaster

Genome ◽

10.1139/g04-060 ◽

2004 ◽

Vol 47 (5) ◽

pp. 849-859 ◽

Cited By ~ 6

Author(s):

Leola Chow ◽

Joel Berube ◽

Alice Fromont ◽

John B Bell

Keyword(s):

Drosophila Melanogaster ◽

Dna Binding ◽

Nuclear Protein ◽

Binding Domain ◽

Dominant Negative ◽

Full Length ◽

Binding Motif ◽

Wing Development ◽

Dna Binding Domain

Scalloped (SD) and Vestigial (VG) proteins physically interact to form a selector complex that activates genes involved in wing development in Drosophila melanogaster. SD belongs to a conserved family of transcription factors containing the TEA/ATTS DNA-binding motif. VG is also a nuclear protein providing the activator function for the SD VG complex. The TEA DNA-binding domain and the VG interacting domain (VID) of SD have been previously identified and described. However, they, and possibly other functional domains of SD, have not been thoroughly characterized in vivo. Herein, transgenic constructs encoding various truncations of SD were used to assess their respective ability to rescue the mutant wing phenotype of two viable sd recessive mutations (sdETX4 and sd58d). The transgenic strains produced were also tested for the ability to induce further sd expression, an ability possessed by full length SD. The functional dissection of SD confirms that specific regions are necessary for wing development and provides further information as to how the SD VG complex functions to promote wing fate. Previous experiments have shown that expression of full length SD can cause a dominant negative wing phenotype. We show that expression of constructs that delete the SD DNA-binding domain can also cause a dominant negative phenotype in a background with either of the two tester sd strains. In contrast, SD constructs that delete the VID have no effect on the wing phenotype in either tester background. Finally, a significant portion of SD at the N-terminal end appears to be dispensable with respect to normal wing development, as this construct behaves the same as full length SD in our assays.Key words: Drosophila melanogaster, wing, scalloped, vestigial, nuclear protein.

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The soluble metal-binding domain of the copper transporter ATP7B binds and detoxifies cisplatin

Biochemical Journal ◽

10.1042/bj20081359 ◽

2009 ◽

Vol 419 (1) ◽

pp. 51-59 ◽

Cited By ~ 46

Author(s):

Nataliya V. Dolgova ◽

Doug Olson ◽

Svetlana Lutsenko ◽

Oleg Y. Dmitriev

Keyword(s):

Metal Binding ◽

Binding Sites ◽

Active Drug ◽

Binding Domain ◽

Copper Binding ◽

Protein Fragment ◽

Copper Transporter ◽

Metal Binding Domain ◽

Cell Expression

Wilson disease ATPase (ATP7B) has been implicated in the resistance of cancer cells to cisplatin. Using a simple in vivo assay in bacterial culture, in the present study we demonstrate that ATP7B can confer resistance to cisplatin by sequestering the drug in its N-terminal metal-binding domain without active drug extrusion from the cell. Expression of a protein fragment containing four N-terminal MBRs (metal-binding repeats) of ATP7B (MBR1–4) protects cells from the toxic effects of cisplatin. One MBR1–4 molecule binds up to three cisplatin molecules at the copper-binding sites in the MBRs. The findings of the present study suggest that suppressing enzymatic activity of ATP7B may not be an effective way of combating cisplatin resistance. Rather, the efforts should be directed at preventing cisplatin binding to the protein.

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Functionally distinct roles for T and Tbx6 during mouse development

Biology Open ◽

10.1242/bio.054692 ◽

2020 ◽

Vol 9 (8) ◽

pp. bio054692

Author(s):

Amy K. Wehn ◽

Deborah R. Farkas ◽

Carly E. Sedlock ◽

Dibya Subedi ◽

Deborah L. Chapman

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcriptional Activity ◽

Es Cells ◽

Binding Domain ◽

Dna Binding Domain ◽

Mouse Development ◽

T Box

ABSTRACTThe mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least in vitro. We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo. We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete in vivo. First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/− TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes in vivo.

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Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

Molecular and Cellular Biology ◽

10.1128/mcb.15.2.861 ◽

1995 ◽

Vol 15 (2) ◽

pp. 861-871 ◽

Cited By ~ 110

Author(s):

K M Catron ◽

H Zhang ◽

S C Marshall ◽

J A Inostroza ◽

J M Wilson ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcriptional Repression ◽

Binding Domain ◽

Specific Cell ◽

Homeodomain Protein ◽

Dna Binding Domain ◽

Dna Binding Sites

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

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