Zinc finger motif for single-stranded nucleic acids? investigations by nuclear magnetic resonance

Nuclear magnetic resonance (NMR) methods have been used to address issues regarding the relevance and feasibility of zinc binding to "zinc finger-like" sequences of the type C-X2-C-X4-H-X4-C [referred to as CCHC or retroviral-type (RT) zinc finger sequences]. One-dimensional (1D) NMR experiments with an 18-residue synthetic peptide containing the amino acid sequence of an HIV-1 RT-zinc finger domain (HIV1-F1) indicate that the sequences are capable of binding zinc tightly and stoichiometrically. 1H-113Cd spin echo difference NMR data confirm that the Cys and His amino acids are coordinated to metal in the 113Cd adduct. The 3D structure of the zinc adduct [Zn(HIV1-F1)] was determined to high atomic resolution by a new NMR-based approach that utilizes 2D-NOESY back-calculations as a measure of the consistency between the structures and the experimental data. Several interesting structural features were observed, including (1) the presence of extensive internal hydrogen bonding, and (2) the similarity of the folding of the first six residues to the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin. Structural constraints associated with conservatively substituted glycines provide further rationale for the physiological relevance of the zinc adduct. Similar NMR and structural results have been obtained for the second HIV-1 RT-zinc finger peptide, Zn(HIV1-F2). NMR studies of the zinc adduct with the NCP isolated directly from HIV-1 particles provide solid evidence that zinc finger domains are formed that are conformationally similar (if not identical) to the peptide structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-resolution structure of an HIV zinc fingerlike domain via a new NMR-based distance geometry approach

A new method is described for determining molecular structures from NMR data. The approach utilizes 2D NOESY back-calculations to generate simulated spectra for structures obtained from distance geometry (DG) computations. Comparison of experimental and back-calculated spectra, including analysis of cross-peak buildup and auto-peak decay with increasing mixing time, provides a quantitative measure of the consistence between the experimental data and generated structures and allows for use of tighter interproton distance constraints. For the first time, the "goodness" of the generated structures is evaluated on the basis of their consistence with the actual experimental data rather than on the basis of consistence with other generated structures. This method is applied to the structure determination of an 18-residue peptide with an amino acid sequence comprising the first zinc fingerlike domain from the gag protein p55 of HIV. This is the first structure determination to atomic resolution for a retroviral zinc fingerlike complex. The peptide [Zn(p55F1)] exhibits a novel folding pattern that includes type I and type II NH-S tight turns and is stabilized both by coordination of the three Cys and one His residues to zinc and by extensive internal hydrogen bonding. The backbone folding is significantly different from that of a "classical" DNA-binding zinc finger. Residues C(1)-F(2)-N(3)-C(4)-G(5)-K(6) fold in a manner virtually identical with the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin; superposition of all main-chain and Cys side-chain atoms of residues C(1)-K(6) of Zn(p55F1) onto residues C(6)-Y(11) and C(39)-V(44) of rubredoxin gives RMSDs of 0.46 and 0.35 A, respectively. The side chains of conservatively substituted Phe and Ile residues implicated in genomic RNA recognition form a hydrophobic patch on the peptide surface.     

Zinc- and sequence-dependent binding to nucleic acids by the N-terminal zinc finger of the HIV-1 nucleocapsid protein: NMR structure of the complex with the Psi-site analog, dACGCC.

The nucleic acid interactive properties of a synthetic peptide with sequence of the N-terminal CCHC zinc finger (CCHC = Cys-X2-Cys-X4-His-X4-Cys; X = variable amino acid) of the human immunodeficiency virus (HIV) nucleocapsid protein, Zn(HIV1-F1), have been studied by 1H NMR spectroscopy. Titration of Zn(HIV1-F1) with oligodeoxyribonucleic acids containing different nucleotide sequences reveals, for the first time, sequence-dependent binding that requires the presence of at least one guanosine residue for tight complex formation. The dynamics of complex formation are sensitive to the nature of the residues adjacent to guanosine, with residues on the 3' side of guanosine having the largest influence. An oligodeoxyribonucleotide with sequence corresponding 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 the bound nucleic acid exists predominantly in a single-stranded, A-helical conformation, and the presence of more than a dozen intermolecular NOE cross peaks enabled three-dimensional modeling of the complex. The nucleic acid binds within a hydrophobic cleft on the peptide surface. This hydrophobic cleft is defined by the side chains of residues Val1, Phe4, Ile12, and Ala13. Backbone amide protons of Phe4 and Ala13 and the backbone carbonyl oxygen of Lys2 that lie within this cleft appear to form hydrogen bonds with the guanosine O6 and N1H atoms, respectively. In addition, the positively charged side chain of Arg14 is ideally positioned for electrostatic interactions with the phosphodiester backbone of the nucleic acid. The structural findings provide a rationalization for the general conservation of these hydrophobic and basic residues in CCHC zinc fingers, and are consistent with site-directed mutagenesis results that implicate these residues as direct participants in viral genome recognition.     

Solution structure of the zinc finger HIT domain in protein FON

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.     

Nucleic acid interactive properties of a peptide corresponding to the N-terminal zinc finger domain of HIV-1 nucleocapsid protein.

An 18-residue peptide (NC-F1) with an amino acid sequence corresponding to the N-terminal zinc finger of human immunodeficiency virus-1 nucleocapsid protein has been shown to bind to nucleic acids by fluorescence and NMR methods. Previously, this peptide has been shown to fold into a defined structure when bound to zinc (Summers et al., 1990). We have used a fluorescent polynucleotide, poly(ethenoadenylic acid), to monitor binding of this peptide to nucleic acids. In the presence of zinc, the peptide had a smaller site size (1.75 nucleotide residues/peptide) than in the absence of the metal ion (2.75). The salt sensitivity of the interaction indicated that two ion pairs are involved in the association of Zn2+ (NC-F1) with polynucleotide, whereas one ion pair is found in the metal-free peptide-nucleic acid complex. Competition experiments with single-stranded DNA (ss DNA) in either the presence or absence of Zn2+ showed that the peptide bound to ss DNA. Using NMR methods, we monitored the binding of a synthetic oligonucleotide, d(TTTGGTTT), to Zn(NC-F1). The hydrophobic residues F2 and I10, which are on the surface of the peptide and have been implicated in viral RNA recognition, were shown to interact with the oligomer. In accord with this observation, analysis of the salt dependence of the polynucleotide-peptide interaction indicates a nonelectrostatic component of about -6 kcal/mol, a value consistent with theoretical estimates of stacking energies of phenylalanine with nucleic acid bases.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Nuclear magnetic resonance structures of the zinc finger domain of human DNA polymerase-alpha

The carboxy terminus of the human DNA polymerase-alpha contains a zinc finger motif. Three-dimensional structures of this motif containing 38 amino acid residues, W L I C E E P T C R N R T R H L P L Q F S R T G P L C P A C M K A T L Q P E, were determined by nuclear magnetic resonance (NMR) spectroscopy. The structures reveal an alpha-helix-like domain at the amino terminus, extending 13 residues from L2 through H15 with an interruption at the sixth residue. The helix region is followed by three turns (H15-L18, T23-L26 and L26-A29), all of which involve proline. The first turn appears to be type III, judging by the dihedral angles. The second and third turns appear to be atypical. A second, shorter helix is formed at the carboxy terminus extending from C30 through L35. A fourth type III turn starting at L35 was also observed in the structure. Proline serves as the third residue of all the turns. Four cysteine residues, two located at the beginning of the helix at the N-terminus and two at the carboxy end, are coordinated to Zn(II), facilitating the formation of a loop. One of the cysteines at the carboxy terminus is part of the atypical turn, while the other is the part of the short helix. These structural features are consistent with the circular dichroism (CD) measurements which indicate the presence of 45% helix, 11% beta turns and 19% non-ordered secondary structures. The zinc finger motif described here is different from those observed for C(4), C(2)H(2), and C(2)HC modules reported in the literature. In particular, polymerase-alpha structures exhibit helix-turn-helix motif while most zinc finger proteins show anti-parallel sheet and helix. Several residues capable of binding DNA, T, R, N, and H are located in the helical region. These structural features imply that the zinc finger motif is most likely involved in binding DNA prior to replication, presumably through the helical region. These results are discussed in the context of other eukaryotic and prokaryotic DNA polymerases belonging to the polymerase B family.     

NMR structure of the complex between the zinc finger protein NCp10 of Moloney murine leukemia virus and the single-stranded pentanucleotide d(ACGCC): comparison with HIV-NCp7 complexes.

The structure of the 56 amino acid nucleocapsid protein NCp10 of retrovirus MoMuLV, which contains a single CX(2)CX(4)HX(4)C-type zinc finger, has been determined previously by NMR. The important role of NCp10 (or NCp7 for HIV-1) in the retroviral life cycle seems mainly related to their preferential binding to single-stranded nucleic acids. We report here the structure of the complex formed between the biologically active (14-53)NCp10 and the oligonucleotide d(ACGCC) in aqueous solution determined by 2D (1)H NMR based methods. The aromatic residue Trp(35) of NCp10 directs nucleic acid complexation as shown by its complete fluorescence quenching upon addition of d(ACGCC). (1)H and (31)P NMR studies support the insertion of Trp(35) between the G(3) and C(4) bases. A total of 577 NOE distance restraints, of which 40 were intermolecular, were used for the structure determination. The zinc finger provides a well-defined surface for the binding of d(ACGCC) through hydrophobic interactions and tryptophan stacking on the guanine. This latter interaction was also observed in the NMR-derived structures of the complexes between NCp7, which contains two successive zinc fingers, and single-stranded DNA and RNA, supporting the proposal for a major role played by aromatic residues of NCp proteins in nucleic acid recognition. Upon binding to the nucleotide a new loop in NCp10 that participates in the intermolecular interaction is formed. Additional interactions provided by positively charged residues surrounding the zinc finger appear necessary for tight binding. The structure of the complex NCp10-d(ACGCC) gives a structural explanation for the loss of virus infectivity following point mutations in the finger domain.     

Alternating zinc fingers in the human male associated protein ZFY: refinement of the NMR structure of an even finger by selective deuterium labeling and implications for DNA recognition.

ZFY, a male-associated Zn-finger protein encoded by the human Y chromosome, exhibits a distinctive two-finger repeat: whereas odd-numbered domains fit a general consensus, even-numbered domains exhibit systematic differences. Do these odd and even sequences encode structurally distinct surfaces for DNA recognition? As a first step toward answering this question, we have recently described the sequential 1H NMR assignment of a representative nonconsensus Zn finger (designated ZFY-6T) based on 2D NMR studies of a 30-residue peptide [Kochoyan, M., Havel, T.F., Nguyen, D.T., Dahl, C.E., Keutmann, H. T., & Weiss, M.A. (1991) Biochemistry 30, 3371-3386]. Initial structural modeling by distance geometry/simulated annealing (DG/SA) demonstrated that this peptide retained the N-terminal beta-hairpin and C-terminal alpha-helix (beta beta alpha motif) observed in consensus Zn fingers. However, the precision of this initial structure was limited by resonance overlap, which led to ambiguities in the assignment of key NOEs in the hydrophobic core. In this paper these ambiguities are resolved by selective deuterium labeling, enabling a refined structure to be calculated by DG/SA and restrained molecular dynamics. These calculations provide a detailed view of the hydrophobic core and protein surface, which are analyzed in reference to previously characterized Zn fingers. Variant (even) and consensus (odd) aromatic residues Y10 and F12, shown in an "aromatic swap" analogue to provide equivalent contributions to the hydrophobic core [Weiss, M.A., & Keutmann, H.T. (1990) Biochemistry 29, 9808-9813], nevertheless exhibit striking differences in packing interactions: Y10--but not F12--contributes to a contiguous region of the protein surface defined by putative specificity-determining residues. Alternating surface architectures may have implications for the mechanism of DNA recognition by the ZFY two-finger repeat.     

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.     

Overexpression and purification of single zinc finger peptides of human zinc finger protein ZNF191

ZNF191, a new human zinc finger protein, probably relates to some hereditary diseases and cancers. To obtain structural information of zinc finger domain a convenient method for obtaining milligram quantities of each zinc finger peptide of ZNF191 is necessary. Here, we report an Escherichia coli expression system for rapid and high-level expression of zinc finger 3 and zinc finger 4 of ZNF191. The gene of zinc finger 3 or zinc finger 4 was cloned into pET31b vector to allow expression of single zinc finger peptide as a ketosteroid isomerase (KSI) fusion protein. The KSI-single zinc finger fusion protein was overexpressed in the form of inclusion body, which can be purified by washing several times using buffer solutions, and then be cleaved directly by cyanogen bromide to release single zinc finger peptide. The more than 20mg/L yield of single zinc finger peptide was achieved with more than 95% purity by using YM ultrafiltration membranes. Circular dichroism spectra of these two single zinc finger peptides titrated with Zn(2+) ions demonstrate that they have different secondary structures.     

Greater binding affinity of trivalent antimony to a CCCH zinc finger domain compared to a CCHC domain of kinetoplastid proteins

It has been reported recently that Sb(III) competes with Zn(II) for its binding to the CCHC zinc finger domain of the HIV-1 NCp7 protein, suggesting that zinc finger proteins may be molecular targets for antimony-based drugs. Here, the interaction of Sb(III) with a CCCH zinc finger domain, which is considered to play a crucial role in the biology of kinetoplastid protozoa, has been characterized for the first time. The binding characteristics of Sb(III) were compared between a CCCH-type peptide derived from a kinetoplastid protein and two different CCHC-type zinc finger peptides. The formation of 1 : 1 Zn-peptide and Sb-peptide complexes from the different peptides was demonstrated using circular dichroism, UV absorption, fluorescence spectroscopies and ESI-MS. Titration of the Zn-peptide complexes with SbCl(3) was performed at pH 6 and 7, exploiting the intrinsic fluorescence of the peptides. The differential spectral characteristics of the peptides allowed for competition experiments between the different peptides for binding of Zn(II). The present study establishes that Sb(III) more effectively displaces Zn(II) from the CCCH peptide than CCHC ones, as a result of both the greater stability of the Sb-CCCH complex (compared to Sb-CCHC complexes) and the lower stability of the Zn-CCCH complex (compared to Zn-CCHC complexes). Comparison of the binding characteristics of Sb(III) or Zn(II) between the CCHC-type peptides with different amino acid sequences supports the model that not only the conserved zinc finger motif, but also the sequence of non-conserved amino acids determines the binding affinity of Sb(III) and Zn(II). These data suggest that the interaction of Sb(III) with CCCH-type zinc finger proteins may modulate, or even mediate, the pharmacological action of antimonial drugs.     

Structure of the His44 --> Ala single point mutant of the distal finger motif of HIV-1 nucleocapsid protein: a combined NMR, molecular dynamics simulation, and fluorescence study

The nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers that strongly bind Zn(2+) through coordination of one His and three Cys residues. It has been suggested that NCp7 function is conformation specific since substitution of any of the zinc coordinating residues in the zinc finger motifs leads to subsequent loss of viral infectivity. To further determine the structural requirements necessary for this specific conformation, we investigated by (1)H 2D NMR and molecular dynamics simulations the structure of the distal finger motif of NCp7 in which the zinc coordinating amino acid, His 44, was substituted by a noncoordinating Ala residue. While the fold of the N-terminal part of this mutated peptide was similar to that of the native peptide, an increased lability and significant conformational changes were observed in the vicinity of the His-to-Ala mutation. Moreover, molecular dynamics simulations suggested a mechanism by which the variant peptide can bind zinc ion even though one zinc-coordinating amino acid was lacking. Using the fluorescence of the naturally occurring Trp37 residue, the binding affinity of the variant peptide to the (TG)(3) model oligonucleotide was found to be decreased by about 2 orders of magnitude with respect with the native peptide. Modeling of the DNA:NCp7 complex using structures of the variant peptide suggests that the residues forming a hydrophobic cleft in the native protein are improperly oriented for efficient DNA binding by the variant peptide.     

The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.     

N-Terminal domain of HTLV-I integrase. Complexation and conformational studies of the zinc finger.

The HTLV-I integrase N-terminal domain [50-residue peptide (IN50)], and a 35-residue truncated peptide formed by residues 9-43 (IN35) have been synthesized by solid-phase peptide synthesis. Formation of the 50-residue zinc finger type structure through a HHCC motif has been proved by UV-visible absorption spectroscopy. Its stability was demonstrated by an original method using RP-HPLC. Similar experiments performed on the 35-residue peptide showed that the truncation does not prevent zinc complex formation but rather that it significantly influences its stability. As evidenced by CD spectroscopy, the 50-residue zinc finger is unordered in aqueous solution but adopts a partially helical conformation when trifluoroethanol is added. These results are in agreement with our secondary structure predictions and demonstrate that the HTLV-I integrase N-terminal domain is likely to be composed of an helical region (residues 28-42) and a beta-strand (residues 20-23), associated with a HHCC zinc-binding motif. Size-exclusion chromatography showed that the structured zinc finger dimerizes through the helical region.