Combining structure based design with phage display to create new Cys ( 2 ) His ( 2 ) zinc finger … (2000 Oct)
combining structure based design with phage display to create new Cys ( 2 ) His ( 2 ) zinc finger dimers . background : several strategies have been reported for the design and selection of novel DNA binding proteins . Most of these studies have used Cys ( 2 ) His ( 2 ) zinc finger proteins as a framework , and have focused on constructs that bind DNA in a manner similar to zif268 , with neighboring fingers connected by a canonical ( krüppel type ) linker . This linker does not seem ideal for larger constructs because only modest improvements in affinity are observed when more than three fingers are connected in this manner . Two strategies have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site : connecting sets of fingers using linkers ( covalent ) , or assembling sets of fingers using dimerization domains ( non covalent ) . results : using a combination of structure based design and phage display , we have developed a new dimerization system for Cys ( 2 ) His ( 2 ) zinc fingers that allows the assembly of more than three fingers on a desired target site . Zinc finger constructs employing this new dimerization system have high affinity and good specificity for their target sites both in vitro and in vivo . constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc …

Identification of DNA recognition sequences and protein interaction domains of the multiple Zn finger protein Roaz. (1998 Nov)
identification of DNA recognition sequences and protein interaction domains of the multiple Zn finger protein Roaz . Roaz , a rat C2H2 zinc finger protein , plays a role in the regulation of olfactory neuronal differentiation through its interaction with the Olf 1 / EBF transcription factor family . An additional role for the Roaz / Olf 1 / EBF heterodimeric protein is suggested by its ability to regulate gene activation at a distinct promoter lacking Olf 1 / EBF binding sites . using an in vitro binding site selection assay ( selex ) , we demonstrate that Roaz protein binds to novel inverted perfect or imperfect repeats of gcaccc separated by 2 bp . We show that Roaz is capable of binding to a canonical consensus recognition sequence with high affinity ( Kd 3 nM ) . analysis of the structural requirement for protein dimerization and DNA binding by Roaz reveals the role of specific zinc finger motifs in the Roaz protein for homodimerization and heterodimerization with the Olf 1 / EBF transcription factor . The DNA binding domain of Roaz is mapped to the N terminal 277 amino acids , containing the first seven zinc finger motifs , which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repeat in a binding site dependent manner . Full length protein can form dimers on both the inverted repeat and direct repeat but not on a single half site . these findings …

Metal and DNA binding properties and mutational analysis of the transcription activating factor , B , of coliphage 186 : … (1997 Aug)
metal and DNA binding properties and mutational analysis of the transcription activating factor , B , of coliphage 186 : a prokaryotic C4 zinc finger protein . coliphage 186 B is a 72 amino acid protein belonging to the Ogr family of analogous transcription factors present in P2 like phage , which contain a Cys X2 Cys X22 Cys X4 Cys presumptive zinc finger motif . The molecular characterization of these proteins has been hampered by their insolubility , a difficulty overcome in the present study by obtaining B as a soluble cadmium containing derivative ( CdB ) . atomic absorption spectroscopy showed the presence of one atom of cadmium per molecule of purified CdB . The UV absorption spectrum revealed a shoulder at 250 nm , characteristic of CysS Cd ( II ) ligand to metal charge transfer transitions , and the difference absorption coefficient after acidification ( delta epsilon 248 , 24 mM 1 cm 1 ) indicated the presence of a Cd ( Cys S ) 4 center . Gel mobility shift analysis of CdB with a 186 late promoter demonstrated specific DNA binding ( KD , app 3 4 microm ) and the protein was shown to activate transcription in vitro from a promoter reporter plasmid construct . The B DNA binding site was mapped by gel shift and dnaase I cleavage protection experiments to an area between 70 and 43 relative to the transcription start site , coincident with the consensus sequence , gttgt N8 …

Variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins. (1997 Jul)
variations of the C2H2 zinc finger motif in the yeast genome and classification of yeast zinc finger proteins . The prosite pattern Zinc finger C2H2 was extended to permit the detection of all C2H2 zinc fingers and their parent proteins in the recently completed sequence of the yeast genome . additionally , a new computer program was written that extracts other zinc binding motifs ( non C2H2 fingers ) , overlapping with the classical zinc finger pattern , from the found set of yeast C2H2 fingers . The complete and correct detection of all fingers is a prerequisite for the classification of the yeast zinc finger proteins in functional terms . The detected 53 yeast C2H2 zinc finger proteins do not contain finger clusters with 10 or more repeats , as is frequently found in higher eukaryotes . Only three proteins contain four or more fingers in a cluster . moreover , nearly all 27 yeast proteins with tandem arrays of two or three finger domains can be classified into nine subgroups with high sequence conservation in their finger clusters , in particular of their DNA recognition helices . these results and application of the recently elaborated finger / DNA recognition rules suggest that the yeast proteins belonging to the same subgroup may recognize identical or very similar DNA sites .

FYVE finger proteins as effectors of phosphatidylinositol 3 phosphate. (1999 Jun)
FYVE finger proteins as effectors of phosphatidylinositol 3 phosphate . phosphatidylinositol 3 phosphate ( ptdins ( 3 ) P ) , generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3 kinase ( PI 3 kinase ) , plays an essential role in intracellular membrane traffic . The underlying mechanism is still not understood in detail , but the recent identification of the FYVE finger as a protein domain that binds specifically to ptdins ( 3 ) P provides a number of potential effectors for ptdins ( 3 ) P . The FYVE finger ( named after the first letter of the four proteins containing it ; fab1p , YOTB , vac1p and EEA1 ) is a double zinc binding domain that is conserved in more than 30 proteins from yeast to mammals . It is found in several proteins involved in intracellular traffic , and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins . The interaction of FYVE fingers with ptdins ( 3 ) P may serve three alternative functions : first , to recruit cytosolic FYVE finger proteins to ptdins ( 3 ) P containing membranes ( in concert with accessory molecules ) ; second , to enrich for membrane bound FYVE finger proteins into ptdins ( 3 ) P containing microdomains within the membrane ; and third , to modulate the activity of membrane bound FYVE finger proteins .

Cloning and functional characterization of Roaz , a zinc finger protein that interacts with O / E 1 to regulate … (1997 Jun)
cloning and functional characterization of Roaz , a zinc finger protein that interacts with O / E 1 to regulate gene expression : implications for olfactory neuronal development . We have identified a protein , Rat O / E 1 associated zinc finger protein ( Roaz ) , that plays a role in regulating the temporal and spatial pattern of olfactory neuronal specific gene expression . This protein functions by interacting with the olfactory factor O / E 1 and modulating its transcriptional activity . Roaz , isolated via a yeast two hybrid screen , encoded a protein containing 29 C2H2 zinc fingers of the tfiiia type . The Roaz mRNA was found in brain , eye , olfactory epithelium , spleen , and heart . In situ hybridization data indicated that Roaz was expressed in the basal layer , consisting of neural precursor cells and immature sensory neurons of the olfactory epithelium , but not in the mature receptor cells . We showed that the Roaz protein bound specifically to O / E 1 by using the yeast two hybrid system . The two proteins formed a stable complex in coimmunoprecipitation and in vitro binding assays . introduction of Roaz and O / E 1 into cells containing an olfactory promoter driven luciferase reporter demonstrated that Roaz abolished O / E 1 mediated transcriptional activation . We propose that the function of Roaz is to modulate negatively the transactivational activity of O / E 1 and to act as …

From repression domains to designer zinc finger proteins : a novel strategy of intracellular immunization against HIV. (1996 Oct)
From repression domains to designer zinc finger proteins : a novel strategy of intracellular immunization against HIV . tissue specific gene regulation of eukaryotic organisms is to a large extent mediated by transcription factors that interact with genomic DNA sequences in a sequence specific manner . The purpose of this synopsis is to put forward the potential of designer zinc finger proteins in treating infections of human immunodeficiency virus ( HIV ) . artificial transcription factors containing designer zinc finger structures fused to activator or repressor domains have been designated transcription response modifiers ( TRMs ) . The principle of engineering TRMs has been derived from the analysis of human krüppel type zinc finger genes and their products . Our research efforts encompass two fascinating features that are displayed by the human krüppel type zinc finger protein KOX1 : 1 ) the krüppel type zinc finger domains display rules of sequence specific DNA recognition , and 2 ) the evolutionarily conserved krüppel associated box ( KRAB ) presents one of the strongest transcriptional repressors identified so far in mammalian organisms . The KRAB repressor activity is postulated to be mediated through co repressor molecules , such as silencing mediating protein 1 ( SMP 1 ) . Thus , the structural organization and functional analysis of zinc finger proteins revealed principles of zinc finger transcription factors that are applicable for reducing the viral load in individuals infected with HIV . In this article , a novel concept of generating therapeutic proteins …

A xenopus zinc finger protein that specifically binds dsrna and RNA DNA hybrids. (1997 Sep)
A xenopus zinc finger protein that specifically binds dsrna and RNA DNA hybrids . proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid binding proteins found in nature . We describe a novel zinc finger protein , dsrbp ZFa , isolated by screening an expression library with dsrna . The dsrbp ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C terminal domain . mobility shift experiments demonstrate that dsrbp ZFa binds dsrna and RNA DNA hybrids with nanomolar dissociation constants and in a sequence independent manner . We also show that DNA and single stranded RNA fail to compete with dsrna for binding suggesting dsrbp ZFa prefers to bind an A form helix . using western analyses we have localized dsrbp ZFa primarily to the nucleus of xenopus laevis oocytes . The identification of dsrbp ZFa provides the first example of a zinc finger protein that is specific for dsrna . In addition , dsrbp ZFa does not contain the previously described dsrna binding motif , suggesting certain zinc fingers may provide an alternative way to recognize the A form helix .

Effect of redox conditions on the DNA binding efficiency of the retinoic acid receptor zinc finger. (1998 Dec)
effect of redox conditions on the DNA binding efficiency of the retinoic acid receptor zinc finger . retinoic acid and its derivatives are involved in many important biological processes . In the present study , we have shown that the DNA binding domain of the retinoic acid receptor , which contains two zinc fingers with the Zn ( II ) tetrahedrally coordinated by four Cys , is susceptible to intracellularly relevant oxidizing agents . In the presence of hydrogen peroxide or hypochlorite , the zinc finger DNA binding activity was abolished in a concentration dependent manner . The loss of DNA binding activity was correlated with the release of Zn ( II ) from the zinc finger motif as a consequence of Zn ( II ) thiolate bond oxidation . A combination of glutathione and Zn ( II ) was able to restore the activity , suggesting that oxidation of the zinc finger by hydrogen peroxide or hypochlorite resulted in the formation of disulfide bonds between the Cys present in the Zn ( II ) binding motif . Our results indicate that in situations of oxidative stress zinc finger containing transcription factors may be particularly susceptible to oxidation , resulting in the disruption of control and regulation of gene expression .

Nuclear transport and phosphorylation of the RNA binding xenopus zinc finger protein XFG 5 1. (1993 Mar)
nuclear transport and phosphorylation of the RNA binding xenopus zinc finger protein XFG 5 1 . XFG 5 1 is a krüppel type xenopus zinc finger protein with specific RNA homopolymer binding activity in vitro . In the oocyte , the protein is distributed between nucleus and cytoplasm ; the nuclear fraction , not the cytoplasm , contains phosphorylated isoform ( s ) of XFG 5 1 . In vitro phosphorylation by use of oocyte / egg extracts or purified casein kinase II is specific to the amino terminal portion of the protein . The carboxy terminal zinc finger domain contains a signal sufficient for nuclear transport . overexpression of either full length XFG 5 1 or of the carboxy terminal portion alone , which maintains RNA binding and nuclear import activities , was achieved in xenopus embryos by mRNA injection . This treatment did not result in impaired regulation of development , suggesting that XFG 5 1 functions in a way distinct from the mode of action exemplified in the drosophila zinc finger protein krüppel .

E6 protein of human papillomavirus type 18 binds zinc. (1989 Oct)
E6 protein of human papillomavirus type 18 binds zinc . The E6 open reading frames of human and animal papillomaviruses encode a transforming protein containing a conserved pattern of repeating cysteine doublets ( cys x x cys ) similar to that found in steroid receptor zinc finger proteins . The spacing between the cysteine doublets , however , is twice as long as in any other zinc finger protein . To demonstrate that an E6 protein could indeed bind zinc , we synthesized the human papillomavirus type 18 E6 protein in insect cells with a baculovirus vector and analysed the protein for zinc binding activity by a zinc blot assay . probing of E6 protein blotted to nitrocellulose from SDS polyacrylamide gels with radioactive 65Zn Cl2 demonstrated that it possessed zinc binding activity . reduction of cysteines with DTT prior to the addition of zinc greatly increased the zinc binding activity of blotted E6 protein . competition experiments showed that Cd2 and Co2 were more effective competitors for zinc binding than Mg2 or Ca2 , indicating that zinc atoms may be tetrahedrally coordinated in E6 zinc complexes . We mapped zinc binding protein domains by proteolytic cleavage and demonstrated that a small 4kDa fragment of the protein retained zinc binding activity , consistent with a model of individual 29 amino acid zinc binding fingers .

Solution structure of the focal adhesion adaptor pinch LIM1 domain and characterization of its interaction with the integrin linked kinase … (2001 May)
solution structure of the focal adhesion adaptor pinch LIM1 domain and characterization of its interaction with the integrin linked kinase ankyrin repeat domain . pinch is a recently identified adaptor protein that comprises an array of five LIM domains . pinch functions through LIM mediated protein protein interactions that are involved in cell adhesion , growth , and differentiation . The LIM1 domain of pinch interacts with integrin linked kinase ( ILK ) , thereby mediating focal adhesions via a specific integrin / ILK signaling pathway . We have solved the NMR structure of the pinch LIM1 domain and characterized its binding to ILK . LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine rich protein and cysteine rich intestinal protein families with CCHC and CCCC zinc finger types . Gel filtration and NMR experiments demonstrated a 1 : 1 complex between pinch LIM1 and the ankyrin repeat domain of ILK . A chemical shift mapping experiment identified regions in pinch LIM1 that are important for interaction with ILK . comparison of surface features between pinch LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins .

Molecular cloning and expression of a leishmania major gene encoding a single stranded DNA binding protein containing nine CCHC zinc … (1993 Jul)
molecular cloning and expression of a leishmania major gene encoding a single stranded DNA binding protein containing nine CCHC zinc finger motifs . The genes encoding GP63 , the major surface glycoprotein of the protozoan parasite leishmania , are highly conserved across diverse species of leishmania both within the protein coding region and in the immediate 5 untranslated region . located between the 3 trans spliced leader acceptor site and the translational initiation codon of the GP63 gene is an area of conserved hexanucleotide direct repeats ( ctcgcc ) which vary in number according to species . To determine whether these repeats represent a site of protein DNA interaction , a leishmania major lambda gt11 expression library was screened with a radiolabeled synthetic oligodeoxynucleotide probe containing multiple ctcgcc repeats to detect clones expressing functional DNA binding proteins . using this approach a gene was isolated which encodes a sequence specific DNA binding protein referred to as hexbp ( hexamer binding protein ) . sequence analysis of the hexbp gene showed that hexbp contains nine cysteine rich motifs which are identical to a consensus sequence known as the CCHC type zinc finger . This motif is present in a number of nucleic acid binding proteins including the nucleocapsid protein of retroviruses . In accordance with the activity exhibited by other proteins containing the CCHC motif , hexbp binds to single stranded nucleic acids as demonstrated by gel mobility shift assays and southwestern blot assays .

A novel human zinc finger protein that interacts with the core promoter element of a TATA box less gene. (1997 May)
A novel human zinc finger protein that interacts with the core promoter element of a TATA box less gene . We describe a novel human cDNA isolated by target site screening of a placental expression library , using as a probe , an essential element of a TATA box less promoter corresponding to a pregnancy specific glycoprotein gene . The cDNA encoded a predicted protein of 290 amino acids , designated core promoter binding protein ( CPBP ) , which has three zinc fingers ( type Cys2 His2 ) at the end of its C terminal domain , a serine / threonine rich central region and an acidic domain lying within the N terminal region . additional sequence analysis and data base searches revealed that only the zinc finger domains are conserved ( 60 80 identity ) in other transcription factors . In cotransfection assays , CPBP increased the transcription from a minimal promoter containing its natural DNA binding site . moreover , a chimeric protein between CPBP and Gal4 DNA binding domain also increased the activity of an heterologous reporter gene containing Gal4 DNA binding sites . The tissue distribution analysis of CPBP mRNA revealed that it is differentially expressed with an apparent enrichment in placental cells . The DNA binding and transcriptional activity of CPBP , in conjunction with its expression pattern , strongly suggests that this protein may participate in the regulation and / or maintenance of the basal expression of PSG and possibly other TATA box …

Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus and plus strand transfer. (2000 Oct)
Zinc finger structures in the human immunodeficiency virus type 1 nucleocapsid protein facilitate efficient minus and plus strand transfer . The nucleocapsid protein ( NC ) of human immunodeficiency virus type 1 ( HIV 1 ) has two zinc fingers , each containing the invariant metal ion binding residues CCHC . recent reports indicate that mutations in the CCHC motifs are deleterious for reverse transcription in vivo . To identify reverse transcriptase ( RT ) reactions affected by such changes , we have probed zinc finger functions in NC dependent RT catalyzed HIV 1 minus and plus strand transfer model systems . Our approach was to examine the activities of wild type NC and a mutant in which all six cysteine residues were replaced by serine ( SSHS NC ) ; this mutation severely disrupts zinc coordination . We find that the zinc fingers contribute to the role of NC in complete tRNA primer removal from minus strand DNA during plus strand transfer . annealing of the primer binding site sequences in plus strand strong stop DNA ( ) ssdna to its complement in minus strand acceptor DNA is not dependent on NC zinc fingers . In contrast , the rate of annealing of the complementary R regions in ( ) ssdna and 3 viral RNA during minus strand transfer is approximately eightfold lower when SSHS NC is used in place of wild type NC . moreover , unlike wild type NC , SSHS NC has only a small stimulatory …

Identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site directed mutagenesis. (2001 Jan)
identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site directed mutagenesis . spermatidal protein TP2 , which appears transiently during stages 12 16 of mammalian spermiogenesis , is a DNA condensing zinc metalloprotein with a preference to GC rich DNA . We have carried out a detailed site directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc . Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues , respectively . The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers . transfection experiments with COS 7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus . The nuclear localization signal in TP2 was identified to be ( 87 ) gkvskrkav ( 95 ) present in the C terminal third of TP2 as a part of an extended NoLS sequence .

Human immunodeficiency virus type 1 nucleocapsid zinc finger mutations cause defects in reverse transcription and integration. (2006 Sep)
human immunodeficiency virus type 1 nucleocapsid zinc finger mutations cause defects in reverse transcription and integration . The nucleocapsid ( NC ) protein from HIV 1 contains two zinc fingers , both of which are necessary for virus replication . This is the first in depth study that presents the effects of nucleocapsid zinc finger substitutions on the kinetics of reverse transcription and integration . Over a 72 h time course of infection , the quantities of viral DNA ( vDNA ) observed with viruses containing either the nucleocapsid his23cys or his44cys mutations were significantly lower than those observed in infections with virus containing wild type NC . In addition , the kinetics of vDNA formation and loss were significantly different from wild type . The kinetic profiles observed indicated reduced vDNA stability , as well as defects in reverse transcription and integration . overall , the defect in integration was much more pronounced than the reverse transcription defects . This suggests that the principal reason for the replication defectiveness of these mutant viruses is impairment of integration , and thus demonstrates the critical importance of NC in HIV 1 infection .

Partial nucleotide sequence and chromosomal localization of a bovine zinc finger gene znf164. (1995 Jul)
partial nucleotide sequence and chromosomal localization of a bovine zinc finger gene znf164 . A clone carrying an open reading frame coding for a novel zinc finger protein of the krüppel family was isolated from a bovine genomic library and designated ZNF 164 ( zinc finger protein 164 ) . partial sequencing revealed that it contained at least 13 zinc finger motifs preceded by a lysine rich region of 60 amino acids . The znf164 protein shared approximately 60 similarity with several zinc finger proteins but did not appear to be orthologous with a previously identified gene . using fluorescence in situ hybridization , the znf164 gene was mapped to bovine chromosome band 17q24 .

Two short basic sequences surrounding the zinc finger of nucleocapsid protein ncp10 of moloney murine leukemia virus are critical for … (1993 Apr)
Two short basic sequences surrounding the zinc finger of nucleocapsid protein ncp10 of moloney murine leukemia virus are critical for RNA annealing activity . The 56 amino acid nucleocapsid protein ( ncp10 ) of moloney murine leukemia virus , contains a cysx2cysx4hisx4cys zinc finger flanked by basic residues . In vitro ncp10 promotes genomic RNA dimerization , a process most probably linked to genomic RNA packaging , and replication primer tRNA ( Pro ) annealing to the initiation site of reverse transcription . To characterize the amino acid sequences involved in the various functions of ncp10 , we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N or C terminus with or without the zinc finger domain . In the latter case , the two parts of the protein were linked by a glycine glycine spacer . The in vitro studies of these peptides show that nucleic acid annealing activities of ncp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues . Thus , deletion of 11R or 49prpqt , of the fully active 29 residue peptide 11rqggerrrsqldrdggkkprgprgprpqt53 leads to a complete loss of ncp10 activity . therefore it is proposed that in ncp10 , the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger .

Transition metals modulate DNA protein interactions of SP1 zinc finger domains with its cognate target site. (1991 Jun)
transition metals modulate DNA protein interactions of SP1 zinc finger domains with its cognate target site . The metal free apoprotein of recombinant human transcription factor SP1 was used in metal reconstitution experiments to study the importance of zinc in facilitating DNA binding of zinc finger proteins . Our functional analysis indicates that several transition metals are capable of modulating DNA protein interactions of zinc finger domains with their cognate DNA target sites . excess or deficiency of divalent zinc , or the presence of transition metals , such as divalent cadmium , cobalt , copper , manganese and nickel impair DNA binding of zinc reconstituted SP1 . In addition , functionally active SP1 protein can be obtained by metal reconstitutions in absence of zinc ( II ) by presence of cadmium ( II ) and cobalt ( II ) , to lesser extents by presence of nickel ( II ) or manganese ( II ) chloride . This study indicates that zinc might play a functional role in regulating DNA protein interactions of zinc finger proteins in vivo . It is postulated that fluctuating divalent zinc alone or transition metals bound to cellular components might form mixed ligand complexes that alter the zinc finger protein conformation and impair DNA binding .