Structural characterization of a 39-residue synthetic peptide containing the two zinc binding domains from the HIV-1 p7 nucleocapsid protein by CD and NMR spectroscopy

A 39-residue peptide (p7-DF) containing the two zinc binding domains of the p7 nucleocapsid protein was prepared by solid-phase peptide synthesis. The solution structure of the peptide was characterized using circular dichroic and nuclear magnetic resonance spectroscopy in both the presence and absence of zinc ions. Circular dichroic spectroscopy indicates that the peptide exhibits a random coil conformation in the absence of zinc but appears to form an ordered structure in the presence of zinc. Two-dimensional nuclear magnetic resonance spectroscopy indicates that the two zinc binding domains within the peptide form stable, but independent, units upon the addition of 2 equivalents of ZnCl2 per equivalent of peptide. Structure calculations on the basis of nuclear Overhauser (NOE) data indicate that the two zinc binding domains have the same polypeptide fold within the errors of the coordinates (approximately 0.5 A for the backbone atoms, the zinc atoms and the coordinating cysteine and histidine ligands). The linker region (Arg17-Gly23) is characterized by a very limited number of sequential NOEs and the absence of any non-sequential NOEs suggest that this region of polypeptide chain is highly flexible. The latter coupled with the occurrence of a large number of basic residues (four out of seven) in the linker region suggests that it may serve to allow adaptable positioning of the nucleic acid recognition sequences within the protein.     

Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.     

Cooperative, non-specific binding of a zinc finger peptide to DNA.

The DNA binding and structural properties of Xfin-31 (Lee, M.S., Gippert, G.P., Soman, K.V., Case, D.A. and Wright, P.E., 1989, Science 245, 635-637), a twenty five amino acid zinc finger peptide, in the reduced, oxidized and zinc complex forms, as well as the fourteen residue helical segment of the zinc finger (residues 12-25) have been compared using affinity coelectrophoresis (ACE) and circular dichroism (CD) spectroscopy. The zinc complex and oxidized peptides bind cooperatively to DNA although the cooperativity factor, omega, is more than 15-fold greater for the zinc complex. The reduced peptide in the absence of zinc and the helical segment do not bind cooperatively (omega = 1). Hence, the binding constant for singly contiguous sites (K omega) ranges over 100-fold for the various peptides even though the intrinsic binding constants (K) are similar. An increase in binding order and affinity for the other forms of Xfin-31 is correlated with an increasing similarity of the CD spectrum to that of the Xfin-31 zinc complex. The surprising DNA binding activity of the oxidized peptide may result from hydrophobic interactions between the amino-terminal loop formed by the Cys3-Cys6 disulfide bond and conserved hydrophobic residues in the carboxyl-terminal segment. Xfin-31 may be a particularly useful model for studying several poorly understood aspects of cooperative, non-specific DNA binding since it is small, has a stable, well-defined structure, and structures of zinc fingers bound to DNA have been determined.     

Two distinct binding modes of a protein cofactor with its target RNA

Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.     

Structure of the p53 transactivation domain in complex with the nuclear receptor coactivator binding domain of CREB binding protein

The activity and stability of the tumor suppressor p53 are regulated by interactions with key cellular proteins such as MDM2 and CBP/p300. The transactivation domain (TAD) of p53 contains two subdomains (AD1 and AD2) and interacts directly with the N-terminal domain of MDM2 and with several domains of CBP/p300. Here we report the NMR structure of the full-length p53 TAD in complex with the nuclear coactivator binding domain (NCBD) of CBP. Both the p53 TAD and NCBD are intrinsically disordered and fold synergistically upon binding, as evidenced by the observed increase in helicity and increased level of dispersion of the amide proton resonances. The p53 TAD folds to form a pair of helices (denoted Pα1 and Pα2), which extend from Phe19 to Leu25 and from Pro47 to Trp53, respectively. In the complex, the NCBD forms a bundle of three helices (Cα1, residues 2066-2075; Cα2, residues 2081-2092; and Cα3, residues 2095-2105) with a hydrophobic groove into which p53 helices Pα1 and Pα2 dock. The polypeptide chain between the p53 helices remains flexible and makes no detectable intermolecular contacts with the NCBD. Complex formation is driven largely by hydrophobic contacts that form a stable intermolecular hydrophobic core. A salt bridge between D49 of p53 and R2105 of NCBD may contribute to the binding specificity. The structure provides the first insights into simultaneous binding of the AD1 and AD2 motifs to a target protein.     

The unique N‐terminal zinc finger of synaptotagmin‐like protein 4 reveals FYVE structure

Synaptotagmin‐like protein 4 (Slp4), expressed in human platelets, is associated with dense granule release. Slp4 is comprised of the N‐terminal zinc finger, Slp homology domain, and C2 domains. We synthesized a compact construct (the Slp4N peptide) corresponding to the Slp4 N‐terminal zinc finger. Herein, we have determined the solution structure of the Slp4N peptide by nuclear magnetic resonance (NMR). Furthermore, experimental, chemical modification of Cys residues revealed that the Slp4N peptide binds two zinc atoms to mediate proper folding. NMR data showed that eight Cys residues coordinate zinc atoms in a cross‐brace fashion. The Simple Modular Architecture Research Tool database predicted the structure of Slp4N as a RING finger. However, the actual structure of the Slp4N peptide adopts a unique C₄C₄‐type FYVE fold and is distinct from a RING fold. To create an artificial RING finger (ARF) with specific ubiquitin‐conjugating enzyme (E2)‐binding capability, cross‐brace structures with eight zinc‐ligating residues are needed as the scaffold. The cross‐brace structure of the Slp4N peptide could be utilized as the scaffold for the design of ARFs.     

Biliary anionic peptide fraction/calcium binding protein inhibits apolipoprotein A-I-mediated cholesterol efflux from cultured cells

The ABC transporter ABCA1 has been implicated to control cholesterol efflux in a variety of cell types including macrophages, fibroblasts, and intestinal epithelial cells. In this study we have investigated whether the 6-kD protein anionic peptide fraction/calcium binding protein (APF/CBP) which has homology to apolipoprotein AI may regulate efflux mediated by lipoproteins. APF/CBP was purified from T-tube bile by ultracentrifugation and preparative reversed phase HPLC. Cholesterol efflux to a variety of acceptors was determined using cultured fibroblasts from controls and patients with Tangiers disease. APF/CBP (0.1 to 2.4 microg/ml) inhibited ApoA-1 (2 microg/ml) mediated cholesterol efflux from normal fibroblasts in a dose dependent manner but had no effect on aspecific efflux to methyl-beta-cyclodextrin or phosphatidylcholine liposomes. In ABCA1 deficient fibroblasts no effect of APF/CBP on efflux was seen. We conclude that APF/CBP specifically interferes with ApoA-I mediated cholesterol trafficking. We hypothesize that competitive binding to ABCA1 may explain the decreased ApoA-I mediated efflux from fibroblasts.     

Solution structure of human BCL-w: modulation of ligand binding by the C-terminal helix

The structure of human BCL-w, an anti-apoptotic member of the BCL-2 family, was determined by triple-resonance NMR spectroscopy and molecular modeling. Introduction of a single amino acid substitution (P117V) significantly improved the quality of the NMR spectra obtained. The cytosolic domain of BCL-w consists of 8 alpha-helices, which adopt a fold similar to that of BCL-xL, BCL-2, and BAX proteins. Pairwise root meant square deviation values were less than 3 A for backbone atoms of structurally equivalent regions. Interestingly, the C-terminal helix alpha8 of BCL-w folds into the BH3-binding hydrophobic cleft of the protein, in a fashion similar to the C-terminal transmembrane helix of BAX. A peptide corresponding to the BH3 region of the pro-apoptotic protein, BID, could displace helix alpha8 from the BCL-w cleft, resulting in helix unfolding. Deletion of helix alpha8 increased binding affinities of BCL-w for BAK and BID BH3-peptides, indicating that this helix competes for peptide binding to the hydrophobic cleft. These results suggest that although the cytosolic domain of BCL-w exhibits an overall structure similar to that of BCL-xL and BCL-2, the unique organization of its C-terminal helix may modulate BCL-w interactions with pro-apoptotic binding partners.     

Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21

Chitin proteins are commonly found in bacteria that utilize chitin as a source of energy. CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium capable of efficient chitin degradation. When grown on chitin, S. marcescens secretes large amounts of CBP21, along with chitin-degrading enzymes. In an attempt to understand the molecular mechanism of CBP21 action, we have determined its crystal structure at 1.55 angstroms resolution. This is the first structure to be solved of a family 33 carbohydrate-binding module. The structure reveals a "budded" fibronectin type III fold consisting of two beta-sheets, arranged as a beta-sheet sandwich, with a 65-residue "bud" consisting of three short helices, located between beta-strands 1 and 2. Remarkably, conserved aromatic residues that have been suggested previously to play a role in chitin binding were mainly found in the interior of the protein, seemingly incapable of interacting with chitin, whereas the structure revealed a surface patch of highly conserved, mainly hydrophilic residues. The roles of six of these conserved surface-exposed residues (Tyr-54, Glu-55, Glu-60, His-114, Asp-182, and Asn-185) were probed by site-directed mutagenesis and subsequent binding studies. All single point mutations lowered the affinity of CBP21 for beta-chitin, as shown by 3-8-fold increases in the apparent binding constant. Thus, binding of CBP21 to chitin seems to be mediated primarily by conserved, solvent-exposed, polar side chains.     

Examining the zinc binding site of the amyloid-beta peptide.

The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues.     

Solution structure of the dimeric zinc binding domain of the chaperone ClpX

ClpX (423 amino acids), a member of the Clp/Hsp100 family of molecular chaperones and the protease, ClpP, comprise a multimeric complex supporting targeted protein degradation in Escherichia coli. The ClpX sequence consists of an NH2-terminal zinc binding domain (ZBD) and a COOH-terminal ATPase domain. Earlier, we have demonstrated that the zinc binding domain forms a constitutive dimer that is essential for the degradation of some ClpX substrates such as gammaO and MuA but is not required for the degradation of other substrates such as green fluorescent protein-SsrA. In this report, we present the NMR solution structure of the zinc binding domain dimer. The monomer fold reveals that ZBD is a member of the treble clef zinc finger family, a motif known to facilitate protein-ligand, protein-DNA, and protein-protein interactions. However, the dimeric ZBD structure is not related to any protein structure in the Protein Data Bank. A trimer-of-dimers model of ZBD is presented, which might reflect the closed state of the ClpX hexamer.     

Multiconnection of identical zinc finger: implication for DNA binding affinity and unit modulation of the three zinc finger domain

Cys(2)-His(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules. They are expected to elevate the DNA binding affinity and specificity by increasing the number of finger modules. To investigate the relation between the number and the DNA binding affinity of the zinc finger, we have designed the two- to four-finger peptides by connecting the central zinc finger (finger 2) of Sp1 with the canonical linker sequence, Thr-Gly-Glu-Lys-Pro. Gel mobility shift assays reveal that the cognate three- and four-finger peptides, Sp1(zf222) and Sp1(zf2222), strongly bind to the predicted target sequences, but the two-finger peptide, Sp1(zf22), does not. Of special interest is the fact that the dissociation constant for Sp1(zf2222) binding to the target DNA is comparable to that for Sp1(zf222). The methylation interference, DNase I and hydroxyl radical footprintings, and circular permutation analyses demonstrate that Sp1(zf2222) binds to its target site with three successive zinc fingers and the binding of the fourth zinc finger is inhibited by DNA bending induced by the binding of the three-finger domain. The present results strongly indicate that the zinc finger protein binds to DNA by the three-finger domain as one binding unit. In addition, this information provides the basis for the design of a novel multifinger protein with high affinity and specificity for long DNA sequences, such as chromosomal DNAs.