DOCSLIB.ORG

Crystal Structure of the BTB Domain from PLZF

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystal Structure of the BTB Domain from PLZF Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12123–12128, October 1998 Biochemistry Crystal structure of the BTB domain from PLZF K. FARID AHMAD,CHRISTIAN K. ENGEL, AND GILBERT G. PRIVE´* Division of Molecular and Structural Biology, Ontario Cancer Institute, and the Department of Medical Biophysics, University of Toronto, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9 Communicated by David Eisenberg, University of California, Los Angeles, CA, August 19, 1998 (received for review July 9, 1998) ABSTRACT The BTB domain (also known as the POZ (7), we estimate that 5–10% of zinc finger proteins also contain domain) is an evolutionarily conserved protein–protein in- BTB domains. The domain is known to form homomeric and teraction motif found at the N terminus of 5–10% of C2H2-type heteromeric associations with other BTB domains (3), and zinc-finger transcription factors, as well as in some actin- given the large number of BTB domain proteins in the associated proteins bearing the kelch motif. Many BTB pro- genomes of higher eukaryotes, an attractive possibility is that teins are transcriptional regulators that mediate gene expres- the domain generates a combinatorial diversity of complexes sion through the control of chromatin conformation. In the within a family of BTB domain proteins. human promyelocytic leukemia zinc finger (PLZF) protein, The organization of the human promyelocytic leukemia zinc the BTB domain has transcriptional repression activity, di- finger (PLZF) protein is typical of BTB domain proteins: the rects the protein to a nuclear punctate pattern, and interacts 120 amino acid BTB domain is found at the N terminus of the with components of the histone deacetylase complex. The protein, followed by a central region of several hundred amino association of the PLZF BTB domain with the histone deacety- acids, and ending with a series of C2H2 Kruppel-type zinc lase complex provides a mechanism of linking the transcrip- fingers. The PLZF protein is a potent transcriptional repressor tion factor with enzymatic activities that regulate chromatin implicated in embryonic development and in hematopoesis (8, conformation. The crystal structure of the BTB domain of 9). It occurs as a fusion protein with the retinoic acid receptor PLZF was determined at 1.9 Å resolution and reveals a tightly a (RARa) in the rare t(11;17) form of acute promyelocytic intertwined dimer with an extensive hydrophobic interface. leukemia (8). In this acute promyelocytic leukemia, the BTB Approximately one-quarter of the monomer surface area is domain from PLZF is responsible for the dominant negative involved in the dimer intermolecular contact. These features properties of the PLZF-RARa fusion against wild-type are typical of obligate homodimers, and we expect the full- RARa-retinoic acid X receptor (RXRa) function (10). Cells length PLZF protein to exist as a branched transcription from the more common t(15;17) PML-RARa acute promy- factor with two C-terminal DNA-binding regions. A surface- elocytic leukemia can be induced to differentiate with treat- exposed groove lined with conserved amino acids is formed at ment with retinoic acid (RA), but PLZF-RARa cells are the dimer interface, suggestive of a peptide-binding site. This responsive to RA only if potentiated with inhibitors of the groove may represent the site of interaction of the PLZF BTB histone deacetylase complex (11–14). Because the BTB do- domain with nuclear corepressors or other nuclear proteins. main from PLZF can associate with mSin3A, histone deacety- lase 1 (14), SMRT (silencing mediator for retinoid and thyroid The BTB domain (Broad-Complex, Tramtrack, and Bric` a hormone receptors) (13), and N-CoR (nuclear receptor core- brac) (1, 2), also known as POZ (poxvirus and zinc finger) (3), pressor) (12), the PLZF–RARa fusion protein has been is an evolutionarily conserved protein–protein interaction proposed to repress transcription at RA-sensitive sites through domain often found in developmentally regulated transcrip- the BTB-mediated recruitment of the histone deacetylase tion factors. The domain is strongly implicated in the regula- complex (11–14). tion of gene expression through the local control of chromatin Because the domain is widely represented in eukaryotic conformation (4). The domain was first identified in a set of genomes, features common to BTB proteins provide crucial Drosophila and poxvirus genes (5), and examples of BTB hints as to the function of the domain. Punctate nuclear domain genes have since been found in organisms ranging staining patterns are commonly seen in zinc-finger BTB pro- from yeast to man. teins (4), and for ZID, PLZF, and BCL6 (or LAZ3), this has A search of the current publicly available sequence data- been shown to require an intact BTB domain (3, 10, 15). Many bases reveals 56 distinct human BTB entries, of which 22 BTB domain proteins, including Tramtrack, PLZF, and BCL6, correspond to named, full-length genes, whereas the remaining are transcriptional repressors, although some, such as GAGA entries are known only as tentative human consensus (THC) factor, can counteract repression by chromatin remodeling sequences, or expressed sequence tags (EST), (a tabulation of (16). As with other conserved domains, we expect that the these genes can be found at http:yyxtal.oci.utoronto.cayprivey domain carries out a common underyling molecular role, btbtable.html). Approximately two-thirds of the full-length which can be used for a variety of biological functions. Our human BTB genes also encode C2H2 zinc finger modules, structure of the PLZF BTB domain reveals that the BTB whereas approximately one-half of the remaining entries con- domain is a tightly interwound dimer and identifies sites at the tain the kelch motif (4, 6). None of the known human BTB dimer interface, which may be important for BTB–BTB do- genes contain more than one single copy of the domain, main interactions, as well as surface features in the dimer, making it likely that most, if not all, of the known BTB which may be involved in interactions with other proteins. tentative human consensus (THC) and expressed sequence tag (EST) entries correspond to distinct genes. Based on the Abbreviation: PLZF, promyelocytic leukemia zinc finger; 3D, three- projection that there are 300–700 zinc finger proteins in man dimensional; CHESS, Cornell High Energy Synchrotron Source; RA, retinoic acid; RARa, RA receptor a; SMRT, silencing mediator for The publication costs of this article were defrayed in part by page charge retinoid and thyroid hormone receptors. Data deposition: The atomic coordinates have been deposited in the payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Data Bank, Biology Department, Brookhaven National Lab- accordance with 18 U.S.C. §1734 solely to indicate this fact. oratory, Upton, NY 11973 (PDB ID code 1BUO). © 1998 by The National Academy of Sciences 0027-8424y98y9512123-6$2.00y0 *To whom reprint requests should be addressed. e-mail: prive@oci. PNAS is available online at www.pnas.org. utoronto.ca. 12123 Downloaded by guest on October 2, 2021 12124 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 95 (1998) METHODS thionine-substituted protein and the structure was determined by multiwavelength anomalous diffraction phasing (Tables 1 Codons 1–132 from a human PLZF cDNA (gift from J. D. and 2). The structure reveals a tightly intertwined homodimer, Licht, Mount Sinai School of Medicine, New York) were consistent with hydrodynamic studies (22). The dimer can be amplified by PCR and subcloned into the plasmid pET-32(a), described roughly as a prolate ellipsoid with overall dimensions producing an expression vector for an N-terminal thioredoxin of '60 Å by 26 Å by 32 Å. Because of the flattened shape of domain followed by a 6X His tag sequence, a 40-aa linker, and amino acids 1–132 of PLZF. The selenomethionyl-substituted the molecule, most residues are at or near the surface of the protein. The central scaffolding of the protein is made up of a fusion protein was overexpressed in the Escherichia coli me- a b thionine auxotroph strain B834(DE3). After cell lysis and cluster of -helices flanked by short -sheets at both the top protein purification by nickel chelate affinity chromatography, and bottom of the molecule (Figs. 1 and 2). By using the DALI the peak fractions containing the fusion protein were pooled server (25), a search for proteins with similar three- and dialyzed against 100 mM NaCl, 50 mM TriszHCl (pH 7.5), dimensional (3D) structures revealed no significant entries in 2.5 mM CaCl2, and 0.5 mM Tris [2-carboxyethyl]phosphine the Brookhaven Protein Data Bank (PDB). The N terminus of hydrochloride (TCEP), and trypsin was added to the fusion each chain is associated with the main body of the other chain, b protein in digest buffer at a ratio of 1:1000 (wtywt) to remove generating a two-stranded antiparallel -sheet between strand the sequences N-terminal to the BTB domain. Under these b1 of one monomer and strand b59 of the other. (For ease of conditions, the domain was resistant to trypsin treatment for discussion, positions on the symmetry-related molecule will be 24 hr at room temperature. The PLZF BTB domain was labeled with primes. In addition, equivalent symmetry-related purified from the digest mixture by anion exchange chroma- pairs of interactions, such as b1yb59 and b19yb5 will not be tography and size exclusion chromatography. The purified explicitly mentioned.) Helix a6 and the b1yb59-sheet, along protein was concentrated to 8 mgyml in 300 mM NaCl, 50 mM with the symmetry-related elements, form an extended, con- TriszHCl (pH 7.5), and 1 mM TCEP. The final protein cave surface on the underside of the protein dimer. Both chains fragment consists of amino acids 6–132 of PLZF, as confirmed begin and end at the base of the dimer, but because of the by Edman N-terminal amino acid sequencing and ion spray swapped amino termini, the N terminus of one chain is next to mass spectrometry.
Load more

Recommended publications
  • "Protein Quaternary Structure: Subunit&Ndash;Subunit
    Protein Quaternary Secondary article Structure: Subunit–Subunit Article Contents . Introduction Interactions . Quaternary Structure Assembly . Folding and Function Susan Jones, University College, London, England . Protein–Protein Recognition Sites . Concluding Remarks Janet M Thornton, University College, London, England The quaternary structure of proteins is the highest level of structural organization observed in these macromolecules. The multimeric proteins that result from quaternary structure formation involve the association of protein subunits through hydrophobic and electrostatic interactions. Protein quaternary structure has important implications for protein folding and function. Introduction from, other components. Using these definitions, the Proteins are organized into a structural hierarchy. The haemoglobin tetramer (comprised of two a and two b polypeptide chain at the primary structural level comprises polypeptide chains) is defined as an oligomer consisting of a linear, noncovalently linked amino acid residue se- two protomers, each consisting of two monomers, i.e. one a quence. Secondary structure is the level at which the linear and one b polypeptide chain. The definition of a subunit sequences aggregate to form structural motifs such as allows the term to be used for either the a-orb-monomer, helices and sheets. The tertiary structure is formed by or for the ab-protomer. The term multimer is also widely packing of the secondary structural elements into one or used in the literature and is defined here as a protein with a more compact globular domains. In many cases proteins finite number of subunits that need not be identical. are composed of only a single polypeptide chain that has The quaternary nature of some proteins was first tertiary structure as its highest level of organization, e.g.
    [Show full text]
  • Gene Section Review
    Atlas of Genetics and Cytogenetics in Oncology and Haematology OPEN ACCESS JOURNAL INIST-CNRS Gene Section Review XRCC2 (X-ray repair cross complementing 2) Paul R Andreassen, Helmut Hanenberg Division of Experimental Hematology and Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati OH, USA; [email protected] (PRA); Department of Pediatrics III, University Children's Hospital Essen, University Duisburg- Essen, Essen Germany; [email protected] (HH) Published in Atlas Database: November 2017 Online updated version : http://AtlasGeneticsOncology.org/Genes/XRCC2ID334ch7q36.html Printable original version : http://documents.irevues.inist.fr/bitstream/handle/2042/69759/11-2017-XRCC2ID334ch7q36.pdf DOI: 10.4267/2042/69759 This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence. © 2019 Atlas of Genetics and Cytogenetics in Oncology and Haematology Clinically, the only known FA-U patient in the Abstract world exhibits severe congenital abnormalities, but XRCC2 is one of five somatic RAD51 paralogs, all had not developed, by seven years of age, the bone of which have Walker A and B ATPase motifs. marrow failure and cancer that are often seen in Each of the paralogs, including XRCC2, has a patients from other FA complementation groups. function in DNA double-strand break repair by Keywords homologous recombination (HR). However, their Fanconi anemia, Breast Cancer Susceptibility, individual roles are not as well understood as that Tumor Suppressor, Homologous Recombination, of RAD51 itself. DNA Repair, RAD51 Paralog The XRCC2 protein forms a complex (BCDX2) with three other RAD51 paralogs, RAD51B, RAD51C and RAD51D. It is believed that the Identity BCDX2 complex mediates HR downstream of Other names: FANCU BRCA2 but upstream of RAD51, as XRCC2 is HGNC (Hugo): XRCC2 involved in the assembly of RAD51 into DNA damage foci.
    [Show full text]
  • Biochemistry-2018-Hartman.Pdf
    Article Cite This: Biochemistry 2019, 58, 1527−1538 pubs.acs.org/biochemistry Experimental Evaluation of Coevolution in a Self-Assembling Particle Emily C. Hartman,† Marco J. Lobba,† Andrew H. Favor,† Stephanie A. Robinson,† Matthew B. Francis,*,†,‡ and Danielle Tullman-Ercek*,§ † Department of Chemistry, University of California, Berkeley, California 94720-1460, United States ‡ Materials Sciences Division, Lawrence Berkeley National Laboratories, Berkeley, California 94720-1460, United States § Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Technological Institute E136, Evanston, Illinois 60208-3120, United States *S Supporting Information ABSTRACT: Protein evolution occurs via restricted evolutionary paths that are influenced by both previous and subsequent mutations. This effect, termed epistasis, is critical in population genetics, drug resistance, and immune escape; however, the effect of epistasis on the level of protein fitness is less well characterized. We generated and characterized a 6615-member library of all two-amino acid combinations in a highly mutable loop of a virus-like particle. This particle is a model of protein self- assembly and a promising vehicle for drug delivery and imaging. In addition to characterizing the effect of all double mutants on assembly, thermostability, and acid stability, we observed many instances of epistasis, in which combinations of mutations are either more deleterious or more beneficial than expected. These results were used to generate rules governing the effects of multiple mutations on the self-assembly of the virus-like particle. rotein evolution occurs through complex pathways, often have a significant impact on biotechnology in the coming involving nonintuitive leaps between functional var- decades.23,24 To maximize this potential, it is important to P − iants.1 3 These paths include local minima and maxima, in understand how non-native functions can be hindered by which the effect of a given mutation depends entirely on the unanticipated epistatic effects.
    [Show full text]
  • Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis
    cells Review Role of Transglutaminase 2 in Cell Death, Survival, and Fibrosis Hideki Tatsukawa * and Kiyotaka Hitomi Cellular Biochemistry Laboratory, Graduate School of Pharmaceutical Sciences, Nagoya University, Tokai National Higher Education and Research System, Nagoya 464-8601, Aichi, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-52-747-6808 Abstract: Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme catalyzing the crosslink- ing between Gln and Lys residues and involved in various pathophysiological events. Besides this crosslinking activity, TG2 functions as a deamidase, GTPase, isopeptidase, adapter/scaffold, protein disulfide isomerase, and kinase. It also plays a role in the regulation of hypusination and serotonylation. Through these activities, TG2 is involved in cell growth, differentiation, cell death, inflammation, tissue repair, and fibrosis. Depending on the cell type and stimulus, TG2 changes its subcellular localization and biological activity, leading to cell death or survival. In normal unstressed cells, intracellular TG2 exhibits a GTP-bound closed conformation, exerting prosurvival functions. However, upon cell stimulation with Ca2+ or other factors, TG2 adopts a Ca2+-bound open confor- mation, demonstrating a transamidase activity involved in cell death or survival. These functional discrepancies of TG2 open form might be caused by its multifunctional nature, the existence of splicing variants, the cell type and stimulus, and the genetic backgrounds and variations of the mouse models used. TG2 is also involved in the phagocytosis of dead cells by macrophages and in fibrosis during tissue repair. Here, we summarize and discuss the multifunctional and controversial Citation: Tatsukawa, H.; Hitomi, K. roles of TG2, focusing on cell death/survival and fibrosis.
    [Show full text]
  • Dimerization and Domain Swapping in G-Protein-Coupled Receptors: a Computational Study Paul R
    Dimerization and Domain Swapping in G-Protein-Coupled Receptors: A Computational Study Paul R. Gouldson, Ph.D., Christopher Higgs, Ph.D., Richard E. Smith, B.Sc., Mark K. Dean, B.Sc., George V. Gkoutos, M.Sc., and Christopher A. Reynolds, Ph.D. In recent years there has been an increasing number of swapped dimers and contact dimers as the models used were reports describing G protein-coupled receptor (GPCR) restricted to the helical part of the receptor. However, it is dimerization and heterodimerization. However, the evidence proposed that for the purpose of signalling, the domain on the nature of the dimers and their role in GPCR swapped dimer and the corresponding contact dimer are activation is inconclusive. Consequently, we present here a equivalent. The evolutionary trace analysis suggests that review of our computational studies on G protein-coupled every GPCR family and subfamily (for which sufficient receptor dimerization and domain swapping. The studies sequence data is available) has the potential to dimerize described include molecular dynamics simulations on through this common functional site on helices 5 and 6. The receptor monomers and dimers in the absence of ligand, in evolutionary trace results on the G protein are briefly the presence of an agonist, and in the presence of an described and these are consistent with GPCR dimerization. antagonist (or more precisely an inverse agonist). Two In addition to the functional site on helices 5 and 6, the distinct sequence-based approaches to studying protein evolutionary trace analysis identified a second functional interfaces are also described, namely correlated mutation site on helices 2 and 3.
    [Show full text]
  • Systematic Characterization of Pan-Cancer Mutation Clusters
    Research Collection Journal Article Systematic characterization of pan-cancer mutation clusters Author(s): Buljan, Marija; Blattmann, Peter; Aebersold, Ruedi; Boutros, Michael Publication Date: 2018-03-01 Permanent Link: https://doi.org/10.3929/ethz-b-000256182 Originally published in: Molecular Systems Biology 14(3), http://doi.org/10.15252/msb.20177974 Rights / License: Creative Commons Attribution 4.0 International This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Published online: March 23, 2018 Article Systematic characterization of pan-cancer mutation clusters Marija Buljan1,2 , Peter Blattmann1 , Ruedi Aebersold1,3,* & Michael Boutros2,4,5,** Abstract or single point mutations. Eventually, they all lead to changes in the expression levels or to altered functions of cancer driver genes and Cancer genome sequencing has shown that driver genes can often their products. Analysis of different cancer genomics datasets has be distinguished not only by the elevated mutation frequency but further underscored a high degree of heterogeneity in the mutation also by specific nucleotide positions that accumulate changes at a frequency and spectrum among different cancer types (Garraway & high rate. However, properties associated with a residue’s poten- Lander, 2013; Lawrence et al, 2013) and uncovered a long tail of tial to drive tumorigenesis when mutated have not yet been low-frequency driver mutations (Garraway & Lander, 2013). As a systematically investigated. Here, using a novel methodological corollary, in spite of the great progress in charting mutational events approach, we identify and characterize a compendium of 180 that define different cancer types, the task to distinguish driver and hotspot residues within 160 human proteins which occur with a passenger mutations in an individual genome remains a formidable significant frequency and are likely to have functionally relevant challenge.
    [Show full text]
  • NCCN Guidelines®) Genetic/Familial High-Risk Assessment: Colorectal
    NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) Genetic/Familial High-Risk Assessment: Colorectal Version 3.2017 — October 10, 2017 NCCN.org Continue Version 3.2017, 10/10/17 © National Comprehensive Cancer Network, Inc. 2017, All rights reserved. The NCCN Guidelines® and this illustration may not be reproduced in any form without the express written permission of NCCN®. NCCN Guidelines Version 3.2017 Panel Members NCCN Guidelines Index Table of Contents Genetic/Familial High-Risk Assessment: Colorectal Discussion * Dawn Provenzale, MD, MS/Chair ¤ Þ Michael J. Hall, MD, MS † ∆ Robert J. Mayer, MD † Þ Duke Cancer Institute Fox Chase Cancer Center Dana-Farber/Brigham and Women’s Cancer Center * Samir Gupta, MD/Vice-chair ¤ Amy L. Halverson, MD ¶ UC San Diego Moores Cancer Center Robert H. Lurie Comprehensive Cancer Reid M. Ness, MD, MPH ¤ Center of Northwestern University Vanderbilt-Ingram Cancer Center Dennis J. Ahnen, MD ¤ University of Colorado Cancer Center Stanley R. Hamilton, MD ≠ Scott E. Regenbogen, MD ¶ The University of Texas University of Michigan Travis Bray, PhD ¥ MD Anderson Cancer Center Comprehensive Cancer Center Hereditary Colon Cancer Foundation Heather Hampel, MS, CGC ∆ Niloy Jewel Samadder, MD ¤ Daniel C. Chung, MD ¤ ∆ The Ohio State University Comprehensive Huntsman Cancer Institute at the Massachusetts General Hospital Cancer Center - James Cancer Hospital University of Utah Cancer Center and Solove Research Institute Moshe Shike, MD ¤ Þ Gregory Cooper, MD ¤ Jason B. Klapman, MD ¤ Memorial Sloan Kettering Cancer Center Case Comprehensive Cancer Center/ Moffitt Cancer Center University Hospitals Seidman Cancer Thomas P. Slavin Jr, MD ∆ Center and Cleveland Clinic Taussig David W. Larson, MD, MBA¶ City of Hope Comprehensive Cancer Institute Mayo Clinic Cancer Center Cancer Center Dayna S.
    [Show full text]
  • Receptor-Like Protein Tyrosine Phosphatase Homodimerizes On
    MOLECULAR AND CELLULAR BIOLOGY, Aug. 2000, p. 5917–5929 Vol. 20, No. 16 0270-7306/00/$04.00ϩ0 Copyright © 2000, American Society for Microbiology. All Rights Reserved. Receptor-Like Protein Tyrosine Phosphatase ␣ Homodimerizes on the Cell Surface 1 2 1 GUOQIANG JIANG, † JEROEN DEN HERTOG, AND TONY HUNTER * Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037,1 and Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands2 Received 28 December 1999/Returned for modification 13 March 2000/Accepted 12 May 2000 We reported previously that the N-terminal D1 catalytic domain of receptor protein-tyrosine phosphatase ␣ (RPTP␣) forms a symmetrical, inhibited dimer in a crystal structure, in which a helix-turn-helix wedge element from one monomer is inserted into the catalytic cleft of the other monomer. Previous functional studies also suggested that dimerization inhibits the biological activity of a CD45 chimeric RPTP and the catalytic activity of an isolated RPTP␴ D1 catalytic domain. Most recently, we have also shown that enforced dimerization in- hibits the biological activity of full-length RPTP␣ in a wedge-dependent manner. The physiological significance of such inhibition is unknown, due to a lack of understanding of how RPTP␣ dimerization is regulated in vivo. In this study, we show that transiently expressed cell surface RPTP␣ exists predominantly as homodimers, suggesting that dimerization-mediated inhibition of RPTP␣ biological activity is likely to be physiologically relevant. Consistent with our published and unpublished crystallographic data, we show that mutations in the wedge region of D1 catalytic domain and deletion of the entire D2 catalytic domain independently reduced but did not abolish RPTP␣ homodimerization, suggesting that both domains are critically involved but that neither is essential for homodimerization.
    [Show full text]
  • A Family of C/EBP-Related Proteins Capable of Forming Covalently Hnked Leucine Zipper Dimers in Vitro
    Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press A family of C/EBP-related proteins capable of forming covalently hnked leucine zipper dimers in vitro Simon C. Williams, Carrie A. Cantwell, and Peter F. Johnson ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201 USA Mouse and rat genomic DNA libraries were screened by reduced stringency hybridization with the DNA-binding domain of the c/ebp gene as a probe. Three genes were isolated that encode bZIP DNA-binding proteins (designated CRPI, CRP2, and CRP3) with strong amino acid sequence similarities to the C/EBP-binding domain. CRP2 is identical to the protein described recently by other groups as NF-IL6, LAP, IL-6DBP, and AGP/EBP, whereas CRP1 and CRP3 represent novel proteins. Several lines of evidence indicate that these three proteins, along with C/EBP, comprise a functional family. Each bacterially expressed polypeptide binds to DNA as a dimer with recognition properties that are virtually identical to those of C/EBP. Every member also bears a conserved cysteine residue at or near the carboxyl terminus, immediately following the leucine zipper, that at least in vitro allows efficient disulfide cross-linking between paired zipper helices. We developed a gel assay for covalent dimers to assess leucine zipper specificities among the family members. The results demonstrate that all pairwise combinations of dimer interactions are possible. To the extent that we have examined them, the same heterodimeric complexes can be detected intracellularly following cotransfection of the appropriate pair of genes into recipient cells.
    [Show full text]
  • Resolving Membrane Receptor Multimerization in Live Cells
    RESOLVING MEMBRANE RECEPTOR MULTIMERIZATION IN LIVE CELLS USING TIME RESOLVED FLUORESCENCE METHODS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Megan J. Kaliszewski Klufas December, 2017 RESOLVING MEMBRANE RECEPTOR MULTIMERIZATION IN LIVE CELLS USING TIME RESOLVED FLUORESCENCE METHODS Megan J. Kaliszewski Klufas Dissertation Approved: Accepted: _____________________________ _____________________________ Advisor Department Interim Chair Dr. Adam W. Smith Dr. Christopher J. Ziegler _____________________________ _____________________________ Committee Member Dean of the College Dr. Chrys Wesdemiotis Dr. John C. Green _____________________________ _____________________________ Committee Member Interim Dean of the Graduate School Dr. David S. Perry Dr. Chand K. Midha _____________________________ _____________________________ Committee Member Date Dr. Michael Konopka ______________________________ Committee Member Dr. Mesfin Tsige ii ABSTRACT The cell membrane is a complex environment made up of thousands of molecular components. The dynamic assembly of these components regulates a myriad of cellular functions, but it is difficult to measure in a biologically relevant context. Pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) is a time resolved fluorescence technique that was used to obtain concentration, mobility and co-diffusion (fc) of membrane proteins in live cells. Several lines of evidence support the hypothesis that homo-dimerization (or even oligomerization) facilitates the function of membrane proteins. The goal of this research was to elucidate the dynamic organization and relative affinity of membrane protein-protein interactions. In order to accomplish this goal, a mathematical model was developed to interpret the cross-correlation value obtained from PIE-FCCS and to quantify the dynamic interactions of membrane receptors in a more rigorous way.
    [Show full text]
  • Modulation of the Stability of a Gene-Regulatory Protein Dimer By
    Proc. Natl. Acad. Sci. USA Vol. 86, pp. 7387-7391, October 1989 Biochemistry Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP (catabolite activator protein/Escherichla coli/afflnity chromatography/subunit exchange/ligand binding) ABRAHAM M. BROWN* AND DONALD M. CROTHERS Department of Chemistry, Yale University, New Haven, CT 06511 Contributed by Donald M. Crothers, July 3, 1989 ABSTRACT We describe an experimental approach to the employed to assess the effect ofnatural ligands such as cAMP measurement ofprotein subunit exchange in which biotinylated and DNA. We find that DNA alone destabilizes the CAP subunits mediate attachment of35S-labeled subunits to a strept- dimer, whereas cAMP has a stabilizing effect. These results avidin column as a result of the exchange process. Application lead us to propose that cAMP regulates the gene-activating of the method to Escherichia coli catabolite activator protein activity of CAP by modulating the equilibrium between (CAP) revealed that in the absence of cAMP, the dimerization monomer and dimer forms. equilibrium constant is 3 x 1010 M-', with a dimer lifetime of 300 min. Exchange of CAP subunits is accelerated at least 1000-fold by the presence of nonspecific DNA, under low ionic MATERIALS AND METHODS strength conditions. Catalysis of exchange also occurs at phys- Protein Preparation. Unlabeled CAP was purified from iological ionic conditions. In contrast, physiological concentra- overproducing Escherichia coli cells by affinity chromatog- tions of cAMP stabilize CAP with respect to subunit exchange raphy on cAMP-agarose (11). Radiolabeled CAP was pre- in either the presence or the absence of DNA.
    [Show full text]
  • Light-Activated Protein Interaction with High Spatial Subcellular Confinement
    Light-activated protein interaction with high spatial subcellular confinement Lorena Benedettia,b,c,d, Andrew E. S. Barentineb,e, Mirko Messaa,b,c,d, Heather Wheelera,b,c,d, Joerg Bewersdorfb,e,f,g, and Pietro De Camillia,b,c,d,f,1 aDepartment of Neuroscience, Yale University School of Medicine, New Haven, CT 06510; bDepartment of Cell Biology, Yale University School of Medicine, New Haven, CT 06510; cHoward Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510; dProgram in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT 06510; eDepartment of Biomedical Engineering, Yale University, New Haven, CT 06520; fKavli Institute for Neuroscience, Yale University School of Medicine, New Haven, CT 06510; and gNanobiology Institute, Yale University, West Haven, CT 06516 Contributed by Pietro De Camilli, January 21, 2018 (sent for review August 10, 2017; reviewed by Takanari Inoue and Mark von Zastrow) Methods to acutely manipulate protein interactions at the sub- Upon light excitation, the photosensitive protein undergoes a cellular level are powerful tools in cell biology. Several blue-light- conformational change and interacts with its binding partner. dependent optical dimerization tools have been developed. In Since the photosensitive protein naturally reverts to its ground these systems one protein component of the dimer (the bait) is state over time, the duration of the interaction between the two directed to a specific subcellular location, while the other compo- components depends on the duration of the photostimulation nent (the prey) is fused to the protein of interest. Upon illumina- and on the switch-on and switch-off kinetics of the photorecep- tion, binding of the prey to the bait results in its subcellular tor.
    [Show full text]