NORMAL GENOMIC

• Malgieri G et al.
• The prokaryotic Cys2His2 zinc-finger adopts a novel fold as revealed bythe NMR structure of Agrobacterium tumefaciens Ros DNA-binding domain.
• Proc Natl Acad Sci U S A. 2007; 104: 17341-6
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• The first putative prokaryotic Cys(2)His(2) zinc-finger domain has beenidentified in the transcriptional regulator Ros from Agrobacteriumtumefaciens, indicating that the Cys(2)His(2) zinc-finger domain,originally thought to be confined to the eukaryotic kingdom, could bewidespread throughout the living kingdom from eukaryotic, both animal andplant, to prokaryotic. In this article we report the NMR solutionstructure of Ros DNA-binding domain (Ros87), providing 79 structuralcharacterization of a prokaryotic Cys(2)His(2) zinc-finger domain. The NMRstructure of Ros87 shows that the putative prokaryotic Cys(2)His(2)zinc-finger sequence is indeed part of a significantly larger zinc-bindingglobular domain that possesses a novel protein fold very different fromthe classical fold reported for the eukaryotic classical zinc-finger. TheRos87 globular domain consists of 58 aa (residues 9-66), is arranged in abetabetabetaalphaalpha topology, and is stabilized by an extensive15-residue hydrophobic core. A backbone dynamics study of Ros87, based on(15)N R(1), (15)N R(2), and heteronuclear (15)N-{(1)H}-NOE measurements,has further confirmed that the globular domain is uniformly rigid andflanked by two flexible tails. Mapping of the amino acids necessary forthe DNA binding onto Ros87 structure reveals the protein surface involvedin the DNA recognition mechanism of this new zinc-binding protein domain.

• Santangelo GM
• Glucose signaling in Saccharomyces cerevisiae.
• Microbiol Mol Biol Rev. 2006; 70: 253-82
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• Eukaryotic cells possess an exquisitely interwoven and fine-tuned seriesof signal transduction mechanisms with which to sense and respond to theubiquitous fermentable carbon source glucose. The budding yeastSaccharomyces cerevisiae has proven to be a fertile model system withwhich to identify glucose signaling factors, determine the relevantfunctional and physical interrelationships, and characterize thecorresponding metabolic, transcriptomic, and proteomic readouts. The earlyevents in glucose signaling appear to require both extracellular sensingby transmembrane proteins and intracellular sensing by G proteins.Intermediate steps involve cAMP-dependent stimulation of protein kinase A(PKA) as well as one or more redundant PKA-independent pathways. The finalsteps are mediated by a relatively small collection of transcriptionalregulators that collaborate closely to maximize the cellular rates ofenergy generation and growth. Understanding the nuclear events in thisprocess may necessitate the further elaboration of a new model foreukaryotic gene regulation, called "reverse recruitment." An essentialfeature of this idea is that fine-structure mapping of nucleararchitecture will be required to understand the reception of regulatorysignals that emanate from the plasma membrane and cytoplasm. Completion ofthis task should result in a much improved understanding of eukaryoticgrowth, differentiation, and carcinogenesis.

• Berkovits-Cymet HJ, Amann BT, Berg JM
• Solution structure of a CCHHC domain of neural zinc finger factor-1 andits implications for DNA binding.
• Biochemistry. 2004; 43: 898-903
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• The structure of a CCHHC zinc-binding domain from neural zinc fingerfactor-1 (NZF-1) has been determined in solution though the use of NMRmethods. This domain is a member of a family of domains that have theCys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structuredetermination reveals a novel fold based around a zinc(II) ion coordinatedto three Cys residues and the second of the two conserved His residues.The other His residue is stacked between the metal-coordinated His residueand a relatively conserved aromatic residue. Analysis of His to Glnsequence variants reveals that both His residues are required for theformation of a well-defined structure, but neither is required forhigh-affinity metal binding at a tetrahedral site. The structure suggeststhat a two-domain protein fragment and a double-stranded DNA binding sitemay interact with a common two-fold axis relating the two domains and thetwo half-sites of the DNA-inverted repeat.

• Westman BJ, Perdomo J, Matthews JM, Crossley M, Mackay JP
• Structural studies on a protein-binding zinc-finger domain of Eos revealboth similarities and differences to classical zinc fingers.
• Biochemistry. 2004; 43: 13318-27
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• The oligomerization domain that is present at the C terminus ofIkaros-family proteins and the protein Trps-1 is important for the properregulation of developmental processes such as hematopoiesis. Remarkably,this domain is predicted to contain two classical zinc fingers (ZnFs),domains normally associated with the recognition of nucleic acids. Thepreference for protein binding by these predicted ZnFs is notwell-understood. We have used a range of methods to gain insight into thestructure of this domain. Circular dichroism, UV-vis, and NMR experimentscarried out on the C-terminal domain of Eos (EosC) revealed that the twoputative ZnFs (C1 and C2) are separable, i.e., capable of foldingindependently in the presence of Zn(II). We next determined the structureof EosC2 using NMR spectroscopy, revealing that, although the overall foldof EosC2 is similar to other classical ZnFs, a number of differencesexist. For example, the conformation of the C terminus of EosC2 appears tobe flexible and may result in a major rearrangement of the zinc ligands.Finally, alanine-scanning mutagenesis was used to identify the residuesthat are involved in the homo- and hetero-oligomerization of Eos, andthese results are discussed in the context of the structure of EosC. Thesestudies provide the first structural insights into how EosC mediatesprotein-protein interactions and contributes to our understanding of whyit does not exhibit high-affinity DNA binding.

• Young ET, Kacherovsky N, Cheng C
• An accessory DNA binding motif in the zinc finger protein Adr1 assistsstable binding to DNA and can be replaced by a third finger.
• Biochemistry. 2000; 39: 567-74
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• The DNA binding domain of Adr1, the protein derived from alcoholdehydrogenase regulatory gene 1, is unusual for zinc finger proteins inthat it consists of two classical Cys2His2 zinc fingers and anamino-terminal proximal accessory region termed PAR. PAR is unstructuredin the free protein and becomes structured in the DNA-bound form. Weinvestigated the role of PAR in DNA binding using molecular andbiochemical approaches, and its importance for activation in vivo, usingAdr1-dependent reporter genes. PAR was unimportant for DNA binding when athird finger was added to Adr1, and its importance was diminished but noteliminated by mutations in finger two that increased DNA binding affinity.The kinetic rate constants for three Adr1 proteins containing or lackingPAR were determined by surface plasmon resonance. PAR increased the onrate and decreased the off rate for specific DNA sites for Adr1 containingwild-type fingers one and two. Surprisingly, PAR had no significant effecton the kinetic rate constants when a third finger was present, or whensingle-stranded DNA was used as the substrate for DNA binding. A mutantform of Adr1-F1F2 in which finger 2 makes three base-specific contactswith DNA had a higher affinity for DNA than Adr1 containing three fingers,yet the mutant protein still depended on PAR for optimal binding affinity.The ability to activate transcription in vivo was correlated with a lowdissociation rate, suggesting that stabilizing an activator at thepromoter might be rate-limiting for transcription in vivo. PAR may haveevolved to lend additional stability to DNA-Adr1 complexes encompassingshort binding sites. In addition, PAR may have a role in transcription ata step after DNA binding since deletion of PAR from Adr1 with threefingers decreased activation in vivo but had no effect on DNA bindingkinetics.

• Bowers PM, Schaufler LE, Klevit RE
• A folding transition and novel zinc finger accessory domain in thetranscription factor ADR1.
• Nat Struct Biol. 1999; 6: 478-85
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• The region responsible for sequence-specific DNA binding by thetranscription factor ADR1 contains two Cys2-His2 zinc fingers and anadditional N-terminal proximal accessory region (PAR). The N-terminal(non-finger) PAR is unstructured in the absence of DNA and undergoes afolding transition on binding the DNA transcription target site. We haveused a set of HN-HN NOEs derived from a perdeuterated protein-DNA complexto describe the fold of ADR1 bound to the UAS1 binding site. The PAR formsa compact domain consisting of three antiparallel strands that contact A-Tbase pairs in the major groove. The three-strand domain is a novel foldamong all known DNA-binding proteins. The PAR shares sequence homologywith the N-terminal regions of other zinc finger proteins, suggesting thatit represents a new DNA-binding module that extends the binding repertoireof zinc finger proteins.

• Taylor WE et al.
• Designing zinc-finger ADR1 mutants with altered specificity of DNA bindingto T in UAS1 sequences.
• Biochemistry. 1995; 34: 3222-30
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• Yeast ADR1 contains two Cys2,His2 zinc fingers needed for DNA binding tothe upstream activation sequence UAS1, with bases T5T6G7-G8A9G10 in theADH2 promoter. Potential DNA-contacting amino acid residues at -1, +3, and+6 in the alpha-helical domains of ADR1's fingers one and two includeRHR-RLR; however, the latter finger two residues Leu146 and Arg149 had notproved to be crucial for ADR1 binding, even though Leu146-T6 and Arg149-T5interactions with UAS1 DNA were predicted. We altered Leu146 or Arg149 byPCR cassette mutagenesis, to study ADR1 mutant binding to 16 UAS1 variantsof thymine bases T5 and T6. Mutation of Leu146 to His, making finger two(RLR) like finger one (RHR), decreased binding to wild type UAS1 havingT6, but enhanced its binding strength to sequences having purines G6 orA6, similar to binding seen between finger one's His118 and base A9 ofUAS1. Mutating Leu146 to Lys caused this finger two RKR mutant to bindstrongly to both G6 and T6, possibly by lysine's amine H-bonding to thecarbonyl of guanine or thymine. Specificity of ADR1 for UAS1 with T6 maythus be due to hydrophobic interaction between Leu146 and the T6 methylgroup. ADR1 mutants with either His or Lys in the central +3 residue (146)of zinc finger two, which have Arg149 in the +6 alpha-helical position,bind with UAS1 mutant sequences having G5 very strongly, T5 strongly, A5intermediately, and C5 weakly.(ABSTRACT TRUNCATED AT 250 WORDS)

• Denzer AJ, Nabholz CE, Spiess M
• Transmembrane orientation of signal-anchor proteins is affected by thefolding state but not the size of the N-terminal domain.
• EMBO J. 1995; 14: 6311-7
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• Upon insertion of a signal-anchor protein into the endoplasmic reticulummembrane, either the C-terminal or the N-terminal domain is translocatedacross the membrane. Charged residues flanking the transmembrane domainare important determinants for this decision, but are not necessarilysufficient to generate a unique topology. Using a model protein that isinserted into the membrane to an equal extent in either orientation, wehave tested the influence of the size and the folding state of theN-terminal domain on the insertion process. A small zinc finger domain orthe full coding sequence of dihydrofolate reductase were fused to theN-terminus. These stably folding domains hindered or even prevented theirtranslocation. Disruption of their structure by destabilizing mutationslargely restored transport across the membrane. Translocation efficiency,however, did not depend on the size of the N-terminal domain within arange of 40-237 amino acids. The folding behavior of the N-terminal domainis thus an important factor in the topogenesis of signal-anchor proteins.

• South TL, Summers MF
• Zinc- and sequence-dependent binding to nucleic acids by the N-terminalzinc finger of the HIV-1 nucleocapsid protein: NMR structure of thecomplex with the Psi-site analog, dACGCC.
• Protein Sci. 1993; 2: 3-19
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• The nucleic acid interactive properties of a synthetic peptide withsequence of the N-terminal CCHC zinc finger (CCHC =Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the humanimmunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have beenstudied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) witholigodeoxyribonucleic acids containing different nucleotide sequencesreveals, for the first time, sequence-dependent binding that requires thepresence of at least one guanosine residue for tight complex formation.The dynamics of complex formation are sensitive to the nature of theresidues adjacent to guanosine, with residues on the 3' side of guanosinehaving the largest influence. An oligodeoxyribonucleotide with sequencecorresponding to a portion of the HIV-1 psi-packaging signal, d(ACGCC),forms a relatively tight complex with Zn(HIV1-F1) (Kd = 5 x 10(-6) M).Two-dimensional nuclear Overhauser effect (NOESY) data indicate that thebound nucleic acid exists predominantly in a single-stranded, A-helicalconformation, and the presence of more than a dozen intermolecular NOEcross peaks enabled three-dimensional modeling of the complex. The nucleicacid binds within a hydrophobic cleft on the peptide surface. Thishydrophobic cleft is defined by the side chains of residues Val1, Phe4,Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and thebackbone carbonyl oxygen of Lys2 that lie within this cleft appear to formhydrogen bonds with the guanosine O6 and N1H atoms, respectively. Inaddition, the positively charged side chain of Arg14 is ideally positionedfor electrostatic interactions with the phosphodiester backbone of thenucleic acid. The structural findings provide a rationalization for thegeneral conservation of these hydrophobic and basic residues in CCHC zincfingers, and are consistent with site-directed mutagenesis results thatimplicate these residues as direct participants in viral genomerecognition.

• Berg JM
• Sp1 and the subfamily of zinc finger proteins with guanine-rich bindingsites.
• Proc Natl Acad Sci U S A. 1992; 89: 11109-10