Letting your fingers do the walking.
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Binding Sites↗
Biomedical subjects
J M Berg
Publications and source records attributed to J M Berg.
Lessons from zinc-binding peptides.
Zinc-finger domains are small metal-binding modules that are found in a wide range of gene regulatory proteins. Peptides corresponding to these domains have provided valuable model systems for examining a number of biophysical parameters entirely unrelated to their nucleic acid binding properties. These include the chemical basis for metal-ion affinity and selectivity, thermodynamic properties related to hydrophobic packing and beta-sheet propensities, and constraints on the generation of ligand-binding and potential catalytic sites. These studies have laid the foundation for applications such as the generation of optically detected zinc probes and the design of metal-binding peptides and proteins with desired spectroscopic and chemical properties.
Amino Acid Sequence↗
Centrosymmetric crystals of biomolecules: the racemate method.
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Anti-Bacterial Agents↗
Metal binding properties and secondary structure of the zinc-binding domain of Nup475.
Nup475 is a nuclear zinc-binding protein of unknown function that is induced in mammalian cells by growth factor mitogens. Nup475 contains two tandemly repeated sequences YKTELCX8CX5CX3H (Cys3His repeats) that are thought to be zinc-bindin domains. Similar sequences have been found in a number of proteins from various species of eukaryotes. To determine the metal binding properties and secondary structure of the putative zinc-binding domains of Nup475, we have used synthetic or recombinant peptides that contain one or two domain sequences. The peptide with a single domain bound 1.0 +/- 0.1 equivalents of Co2+, and the peptide with two domains bound 1.7 +/- 0.4 equivalents of Co2+. Both peptides bound Co2+ and Zn2+ with affinities similar to those of classical zinc finger peptides. In each case, the Co2+ complex exhibited strong d-d transitions characteristic of tetrahedral coordination. For structural studies by nuclear magnetic resonance spectroscopy, we used a more soluble two-domain peptide that had a single amino acid substitution in a nonconserved amino acid residue in the second Cys3His repeat. The mutant peptide unexpectedly showed loss of one of its metal binding sites and displayed ordered structure for only the first Cys3His sequence. On the basis of the nuclear magnetic resonance data, we propose a structure for the Nup475 metal-binding domain in which the zinc ion is coordinated by the conserved cysteines and histidine, and the conserved YKTEL motif forms a parallel sheet-like structure with the C terminus of this domain. This structure is unlike that of any previously described class of metal binding domain.
Amino Acid Sequence↗
DNA unwinding induced by zinc finger protein binding.
Zinc finger domains of the Cys2His2 type are found in a large number of eukaryotic proteins. Various proteins containing these domains have been shown to bind specifically to DNA, RNA, and DNA-RNA hybrids. Structural studies of zinc finger protein-DNA complexes have revealed that the DNA molecules are underwound relative to canonical B-form. It has not been clear if zinc finger proteins recognize preexisting underwound conformations of DNA or if they induce such conformations upon binding. We report that the DNA binding domains of Sp1 and several designed zinc finger proteins unwind DNA upon binding. The extent of unwinding is consistent with that observed in zinc finger protein-DNA cocrystal structures. These DNA deformations may be important in determining overall binding affinities as well as influencing binding site preferences. Furthermore, changes in DNA conformation upon zinc finger protein binding may affect protein-protein interactions important for transcriptional regulation and other activities of zinc finger proteins.
Base Sequence↗
The galvanization of biology: a growing appreciation for the roles of zinc.
Zinc ions are key structural components of a large number of proteins. The binding of zinc stabilizes the folded conformations of domains so that they may facilitate interactions between the proteins and other macromolecules such as DNA. The modular nature of some of these zinc-containing proteins has allowed the rational design of site-specific DNA binding proteins. The ability of zinc to be bound specifically within a range of tetrahedral sites appears to be responsible for the evolution of the side range of zinc-stabilized structural domains now known to exist. The lack of redox activity for the zinc ion and its binding and exchange kinetics also may be important in the use of zinc for specific functional roles.
Amino Acid Sequence↗
Water exchange filter with improved sensitivity (WEX II) to study solvent-exchangeable protons. Application to the consensus zinc finger peptide CP-1.
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Alanine↗
Separation of intramolecular NOE and exchange peaks in water exchange spectroscopy using spin-echo filters.
A technique for separating intramolecular NOE and solvent-proton exchange peaks in exchange spectroscopy is demonstrated. This method utilizes the large differences in relaxation and coupling properties of water and macromolecules to separate the two effects. The spin-echo filter consists of a water-frequency selective 90 degrees pulse followed by a spin-echo sequence. If the echo time is sufficiently long, protein resonances (e.g. C alpha H protons) excited by the selective pulse are removed due to their much shorter T2 values and J-coupling evolution. By combining the filter with exchange spectroscopy (EXSY) or water exchange (WEX) filter experiments, exchange peaks can be selectively observed. In this paper the filter is combined with a modified version of the WEX filter (WEX II filter) with 1D and 2D detection and applied to a zinc finger peptide and to staphylococcal nuclease, allowing estimation of the contribution of intramolecular NOEs to the exchange spectra.
Alanine↗
A 2.2 A resolution crystal structure of a designed zinc finger protein bound to DNA.
Considerable recent effort has been devoted to the design and selection of sequence-specific DNA binding proteins based on tandem arrays of Cys2His2 zinc finger domains. While the DNA binding properties of these designed proteins have been studied extensively, the structural basis for site-specific binding has not been examined experimentally. Here we report the crystal structure of a complex between a protein comprised of three consensus-sequence-based zinc finger domains and an oligonucleotide corresponding to a favourable DNA binding site. This structure reveals relatively simple modular interactions and structural adaptations that compensate for differences in contact residue side-chain lengths.
Amino Acid Sequence↗
Serine at position 2 in the DNA recognition helix of a Cys2-His2 zinc finger peptide is not, in general, responsible for base recognition.
The roles of certain amino acids within zinc finger peptides of the Cys2His2 type in determining DNA-binding site specificity were investigated. A variety of three domain peptides designed using a consensus sequence framework were prepared with different potential DNA-contacting residues and tested in a recently developed specificity determination assay. Proteins with recognition helix sequence Q-1S1S2N3L4Q5K6 and QSSDLQK prefer binding subsites 5'-GAA-3' and 5'-(G/T)C(A/G)-3', respectively. Changing the Ser residues in position 2 to Ala in these proteins did not significantly affect the DNA binding site preferences, indicating that Ser residues, which occur frequently in position 2 in known zinc finger proteins are not, in general, responsible for determining binding site preferences. Examination of proteins with recognition helices HSSNLQK and ASSNLQK revealed considerable loss of binding site discrimination throughout the binding subsite. These observations provide further evidence for the importance of residues in positions -1 and 3 in determining DNA binding specificity. Moreover, these results illustrate the effects of changes of one residue in the recognition helix on binding site preferences throughout the recognition subsite.
Amino Acid Sequence↗
Specific DNA-RNA hybrid binding by zinc finger proteins.
Zinc finger proteins of the Cys2His2 type represent a large class of proteins that have been assumed to function by means of specific interactions with DNA. Experiments motivated by structural characteristics of zinc finger protein-DNA complexes revealed that certain zinc finger proteins bound DNA-RNA hybrids with affinities comparable to or greater than those for DNA duplexes. The interactions between the zinc finger proteins and the DNA-RNA hybrids were dependent on which strand was RNA and were sequence-specific. Thus, interactions with DNA-RNA hybrids should be considered with regard to the biological roles of zinc finger proteins.
Amino Acid Sequence↗
A direct comparison of the properties of natural and designed zinc-finger proteins.
BACKGROUND: Zinc-finger proteins of the Cys2His2 type constitute an important family of DNA-binding proteins. Each zinc-finger domain has three residues that are thought to be important in determining DNA binding site specificity. Proteins have been designed previously by combining zinc-finger domains with a fixed sequence framework with different DNA-contacting residues. RESULTS: We compared the DNA-binding properties of the DNA-binding domain from the human transcription factor Sp1, which contains three zinc fingers, with designed proteins in which the sequences of the structural framework were greatly modified but the presumed DNA-contacting residues were retained. Frameworks based on a zinc-finger consensus sequence and on a minimalist sequence consisting largely of alanine residues were studied. The preference for binding to the target sequence, 5'-(G,T)GG G(C,A)G GG(G,T)-3', was retained in all cases tested. The consensus framework-based protein was found to be superior to the natural one in terms of overall DNA-binding affinity, the degree of sequence discrimination, and the resistance to inactivation by chelating agents. CONCLUSIONS: Our observations provide direct evidence that the residues previously observed to interact with the DNA bases are indeed the most important residues for determining DNA-binding specificity. We have also shown that these domains can tolerate considerable sequence variation while retaining function as well as three-dimensional structure. Finally, they show that framework modification can be used to generate proteins that have normal or enhanced DNA-binding activity but have different metal-binding properties.
Amino Acid Sequence↗
Fibrillin domain folding and calcium binding: significance to Marfan syndrome.
BACKGROUND: Marfan syndrome is a heritable disorder of connective tissue which has been associated with mutations in a gene encoding fibrillin, a 350 kD glycoprotein found in microfibrils. This protein consists of approximately 60 domains, 47 of which have similarity to epidermal growth factor (EGF). The first mutations to be detected were found in two sporadic cases that had identical Arg to Pro changes within one EGF-like domain. Based on sequence features common to 43 of the EGF-like domains, it was proposed that these domains might bind calcium. Through the synthesis and characterization of wild-type and mutated single domain peptides, we examined the structural and calcium-binding properties of an isolated EGF-like domain from fibrillin and the effects of the Arg to Pro sequence change. RESULTS: A peptide corresponding to the thirteenth putative calcium-binding, EGF-like domain of fibrillin (the site of the first detected mutations) was synthesized. This peptide could be easily oxidized and refolded. The structure of this domain was probed using NMR methods, indicating features characteristic of the known structures of EGF-like domains. The domain bound to calcium with moderate affinity (Kd = 0.6 +/- 0.1 mM) with no major changes in structure induced upon calcium binding. A synthetic peptide containing the Arg to Pro mutation was found to be drastically impaired in its ability to fold in vitro. CONCLUSIONS: As predicted, a fibrillin domain forms a calcium-binding, EGF-like module. As the putative calcium-binding sites are found at the amino-terminal end of the modules, we propose that calcium ions may bind in the interfaces between domains, affecting the overall structure of the protein. The Arg to Pro mutation blocks domain folding in vitro, suggesting that lack of proper domain folding in vivo may contribute to the molecular defects responsible for Marfan syndrome.
Amino Acid Sequence↗
Formation of the peroxisome lumen is abolished by loss of Pichia pastoris Pas7p, a zinc-binding integral membrane protein of the peroxisome.
We have cloned and sequenced PAS7, a gene required for peroxisome assembly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mutations that alter conserved residues of the C3HC4 motif abolish PAS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with most pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisomal targeting signal (PTS1) or the type 2 PTS (PTS2). However, while other yeast and mammalian pas mutants accumulate ovoid, vesicular peroxisomal intermediates, loss of Pas7p leads to accumulation of membrane sheets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which is entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the function of Pas7p defines a previously unrecognized step in peroxisome assembly: formation of the peroxisome lumen. Furthermore, because the peroxisomal intermediates in the pas7 delta mutant proliferate in response to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.
Amino Acid Sequence↗
Length-encoded multiplex binding site determination: application to zinc finger proteins.
The screening of combinatorial libraries is becoming a powerful method for identifying or refining the structures of ligands for binding proteins, enzymes, and other receptors. We describe an oligonucleotide library search procedure in which the identity of each member is encoded in the length of oligonucleotides. This encoding scheme allows binding-site preferences to be evaluated via DNA length determination by denaturing gel electrophoresis. We have applied this method to determine the binding-site preferences for 18 Cys2His2 zinc finger domains as the central domain within a fixed context of flanking zinc fingers. An advantage of the method is that the relative affinities of all members of the library can be estimated in addition to simply determining the sequence of the optimal or consensus ligand. The zinc finger domain specificities determined will be useful for modular zinc finger protein design.
Amino Acid Sequence↗
Racemic macromolecules for use in X-ray crystallography.
The ability to chemically synthesize entire macromolecules has allowed the production of racemic mixtures of proteins and oligonucleotides. These racemic mixtures have been used to produce centrosymmetric crystals. Although only modest results have been obtained to date, this technology should be useful for the solution of structures of moderately sized macromolecules and for the generation of electron-density maps with low phase error and model bias.
Crystallography, X-Ray↗
Issues in molecular genetic testing of individuals with suspected early-onset familial Alzheimer's disease.
The identification of mutations in the amyloid precursor protein (APP) gene associated with the presence of early-onset familial Alzheimer disease (AD) raises the possibility of their practical clinical application, at least in some circumstances, in the diagnostic assessment for AD. As a stimulus for discussion, a hypothetical, illustrative case vignette is presented. A 48-year-old man, concerned about recent memory loss and with a family history of early-onset AD, requested testing for the APP717 Val-->Ile mutation, previously identified in his relatives affected with AD. Whether the testing should be undertaken is considered in the context of the current interpretation of potential test results as well as the competency of the individual who requested the test to provide informed consent. Informed consent includes an understanding of the foreseeable risks and benefits associated with disclosure of test results. Although molecular genetic testing in particular individuals, such as the man described herein, could be appropriate, it should not be interpreted to apply in general at this stage to individuals suspected of having AD. In view of a number of caveats, including the genetic heterogeneity of AD, which significantly limits the sensitivity and specificity of the currently available genetic tests, further research and discussion is strongly recommended before widespread introduction of molecular genetic testing for individuals with suspected AD.
Alzheimer Disease↗
Molecular genetic predictive testing for Alzheimer's disease: deliberations and preliminary recommendations.
Forty-one participants representing diverse professional back-grounds attended a workshop on genetic predictive testing for familial Alzheimer's disease (FAD) on January 23, 1993 at Surrey Place Centre in Toronto, Canada. Rapidly emerging molecular genetic findings in AD indicate that predictive testing is now technologically feasible for selected individuals, although defining eligibility criteria remains problematic. Legal, ethical, biomedical, and psychosocial issues related to establishing predictive testing programs for AD were discussed at the workshop. This article reflects these discussions, provides the current biomedical background for them and examines the Huntington's disease (HD) predictive testing experience. Observations concerning molecular genetic predictive testing for AD in light of its genetic heterogeneity and clinical characteristics, such as usual later age of onset than HD, are presented. It is proposed that predictive testing for AD can now be cautiously offered in a research setting primarily according to the recommendations contained within the Ethical Issues Policy Statement on Huntington's Disease Molecular Genetics Predictive Test. However, in their application to AD, some points in the statement are considered to require emphasis, modification, or currently to be of uncertain applicability. This represents an initial step in an on-going process of debate concerning AD that will be required as new advances occur in genetic and clinical research and in bioethics.
Aged↗