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1.
J Mol Biol ; 303(2): 243-53, 2000 Oct 20.
Article in English | MEDLINE | ID: mdl-11023789

ABSTRACT

The TAZ2 (CH3) domain of the transcriptional adapter protein CBP has been implicated in direct functional interactions with numerous cellular transcription factors and viral oncoproteins. The solution structure of the TAZ2 domain of murine CBP has been determined by nuclear magnetic resonance (NMR). The protein adopts a novel helical fold stabilized by three zinc ions, each of which is bound to one histidine and three cysteine ligands in HCCC-type motifs. Each zinc-binding site is formed from the carboxy terminus of an alpha-helix, a short loop, and the amino terminus of the next alpha-helix. A peptide derived from the N-terminal transactivation domain of p53 binds specifically to one face of the TAZ2 domain. The close similarities between the TAZ2 and TAZ1 (CH1 domain of CBP/p300) sequences suggest that both domains will adopt similar three-dimensional structures.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Trans-Activators/chemistry , Trans-Activators/metabolism , Zinc Fingers , Adenovirus E1A Proteins/metabolism , Amino Acid Sequence , Animals , Apoproteins/chemistry , Apoproteins/metabolism , Binding Sites , CREB-Binding Protein , Circular Dichroism , Cysteine/metabolism , Histidine/metabolism , Ligands , Mice , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Solutions , Thermodynamics , Transcriptional Activation , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Zinc/metabolism
2.
J Mol Biol ; 301(2): 491-511, 2000 Aug 11.
Article in English | MEDLINE | ID: mdl-10926523

ABSTRACT

The RNA genome of the human immunodeficiency virus type-1 (HIV-1) contains a approximately 120 nucleotide Psi-packaging signal that is recognized by the nucleocapsid (NC) domain of the Gag polyprotein during virus assembly. The Psi-site contains four stem-loops (SL1-SL4) that possess overlapping and possibly redundant functions. The present studies demonstrate that the 19 residue SL2 stem-loop binds NC with affinity (K(d)=110(+/-50) nM) similar to that observed for NC binding to SL3 (K(d)=170(+/-65) nM) and tighter than expected on the basis of earlier work, suggesting that NC-SL2 interactions probably play a direct role in the specific recognition and packaging of the full-length, unspliced genome. The structure of the NC-SL2 complex was determined by heteronuclear NMR methods using (15)N,(13)C-isotopically labeled NC protein and SL2 RNA. The N and C-terminal "zinc knuckles" (Cys-X(2)-Cys-X(4)-His-X(4)-Cys; X=variable amino acid) of HIV-1 NC bind to exposed guanosine bases G9 and G11, respectively, of the G8-G9-U10-G11 tetraloop, and residues Lys3-Lys11 of the N-terminal tail forms a 3(10) helix that packs against the proximal zinc knuckle and interacts with the RNA stem. These structural features are similar to those observed previously in the NMR structure of NC bound to SL3. Other features of the complex are substantially different. In particular, the N-terminal zinc knuckle interacts with an A-U-A base triple platform in the minor groove of the SL2 RNA stem, but binds to the major groove of SL3. In addition, the relative orientations of the N and C-terminal zinc knuckles differ in the NC-SL2 and NC-SL3 complexes, and the side-chain of Phe6 makes minor groove hydrophobic contacts with G11 in the NC-SL2 complex but does not interact with RNA in the NC-SL3 complex. Finally, the N-terminal helix of NC interacts with the phosphodiester backbone of the SL2 RNA stem mainly via electrostatic interactions, but does not bind in the major groove or make specific H-bonding contacts as observed in the NC-SL3 structure. These findings demonstrate that NC binds in an adaptive manner to SL2 and SL3 via different subsets of inter and intra-molecular interactions, and support a genome recognition/packaging mechanism that involves interactions of two or more NC domains of assembling HIV-1 Gag molecules with multiple Psi-site stem-loop packaging elements during the early stages of retrovirus assembly.


Subject(s)
Gene Products, gag/chemistry , Genome, Viral , HIV-1/chemistry , RNA, Viral/chemistry , Calorimetry , HIV-1/genetics , Magnetic Resonance Spectroscopy , Models, Molecular , Nucleic Acid Conformation , Protein Binding , Virus Assembly
3.
J Mol Biol ; 299(1): 145-56, 2000 May 26.
Article in English | MEDLINE | ID: mdl-10860728

ABSTRACT

The genome of the human immunodeficiency virus type-1 (HIV-1) contains a stretch of approximately 120 nucleotides known as the psi-site that is essential for RNA packaging during virus assembly. These nucleotides have been proposed to form four stem-loops (SL1-SL4) that have both independent and overlapping functions. Stem-loop SL2 is important for efficient recognition and packaging of the full-length, unspliced viral genome, and also contains the major splice-donor site (SD) for mRNA splicing. We have determined the structure of the 19-residue SL2 oligoribonucleotide by heteronuclear NMR methods. The structure is generally consistent with the most recent of two earlier secondary structure predictions, with residues G1-G2-C3-G4 and C6-U7 forming standard Watson Crick base-pairs with self-complementary residues C16-G17-C18-C19 and A12-G13, respectively. However, residue A15, which is located near the center of the stem, does not form a predicted bulge, and residues A5 and U14 do not form an expected Watson-Crick base-pair. Instead, these residues form a novel A5-U14-A15 base-triple that appears to be stabilized by hydrogen bonds from A15-H61 and -H62 to A5-N1 and U14-O2, respectively; from A5-H61 to U14-O2, and from C16-H42 to U14-O2'. A kink in the backbone allows the aromatic rings of the sequential U14-A15 residues to be approximately co-planar, adopting a stable "platform motif" that is structurally similar to the A-A (adenosine) platforms observed in the P4-P6 ribozyme domain of the Tetrahymena group I intron. Platform motifs generally function in RNA by mediating long-range interactions, and it is therefore possible that the A-U-A base-triple platform mediates long-range interactions that either stabilize the psi-RNA or facilitate splicing and/or packaging. Residue G8 of the G8-G9-U10-G11 tetraloop is stacked above the U7-A12 base-pair, and the remaining tetraloop residues are disordered and available for potential interactions with either other RNA or protein components.


Subject(s)
HIV-1/genetics , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , RNA, Viral/chemistry , RNA, Viral/genetics , Virus Assembly/genetics , Animals , Base Pairing/genetics , Base Sequence , Hydrogen Bonding , Introns/genetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Oligoribonucleotides/chemistry , Oligoribonucleotides/genetics , Oligoribonucleotides/isolation & purification , Oligoribonucleotides/metabolism , Protons , RNA Stability , RNA, Viral/isolation & purification , RNA, Viral/metabolism , Tetrahymena/genetics , Thermodynamics
4.
Biochemistry ; 39(7): 1604-12, 2000 Feb 22.
Article in English | MEDLINE | ID: mdl-10677209

ABSTRACT

The nucleocapsid protein (NC) from the mouse mammary tumor virus (MMTV) has been overexpressed in Escherichia coli and purified to homogeneity for structural studies by nuclear magnetic resonance (NMR) spectroscopy. The protein contains two copies of a conserved zinc-coordinating "CCHC array" or "zinc knuckle" motif common to the nucleocapsid proteins of nearly all known retroviruses. The residues comprising and adjacent to the zinc knuckles were assigned by standard two-dimensional (1)H and three-dimensional (1)H-(15)N NMR methods; the rotational dynamic properties of the protein were determined from (15)N relaxation experiments, and distance restraints derived from the nuclear Overhauser effect (NOE) data were used to calculate the three-dimensional structure. The (1)H-(1)H NOE and (15)N relaxation data indicate that the two zinc knuckles do not interact with each other, but instead behave as independently folded domains connected by a flexible 13-residue linker segment. The proximal zinc knuckle folds in a manner that is essentially identical to that observed previously for the two zinc knuckles of the human immunodeficiency virus type 1 nucleocapsid protein and for the moloney murine leukemia virus nucleocapsid zinc knuckle domain. However, the distal zinc knuckle of MMTV NC exhibits a rare three-dimensional fold that includes an additional C-terminal beta-hairpin. A similar C-terminal reverse turn-like structure was observed recently in the distal zinc knuckle of the Mason-Pfizer monkey virus nucleocapsid protein [Gao, Y., et al. (1998) Protein Sci. 7, 2265-2280]. However, despite a high degree of sequence homology, the conformation and orientation of the beta-hairpin in MMTV NC is significantly different from that of the reverse turn in MPMV NC. The results support the conclusion that structural features of NC zinc knuckle domains can vary significantly among the different genera of retroviridae, and are discussed in terms of the recent and surprising discovery that MMTV NC can facilitate packaging of the HIV-1 genome in chimeric MMTV mutants.


Subject(s)
Mammary Tumor Virus, Mouse/chemistry , Nucleocapsid Proteins/chemistry , Peptide Fragments/chemistry , Protein Folding , Zinc Fingers , Amino Acid Sequence , Animals , Crystallography, X-Ray , Humans , Mice , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Spectrum Analysis , Thermodynamics
5.
J Mol Biol ; 279(3): 633-49, 1998 Jun 12.
Article in English | MEDLINE | ID: mdl-9641983

ABSTRACT

The HIV-1 nucleocapsid protein (NC) contains two CCHC-type zinc knuckle domains that are essential for genome recognition, packaging and infectivity. The solution structure of the protein has been determined independently by three groups. Although the structures of the individual zinc knuckle domains are similar, two of the studies indicated that the knuckles behave as independently folded, non-interacting domains connected by a flexible tether, whereas one study revealed the presence of interknuckle NOE cross-peaks, which were interpreted in terms of a more compact structure in which the knuckles are in close proximity. We have collected multidimensional NMR data for the recombinant, isotopically labeled HIV-1 NC protein, and confirmed the presence of weak interknuckle NOEs. However, the NOE data are not consistent with a single protein conformation. 15N NMR relaxation studies reveal that the two zinc knuckle domains possess different effective rotational correlation times, indicating that the knuckles are not tumbling as a single globular domain. In addition, the 1H NMR chemical shifts of isolated zinc knuckle peptides are very similar to those of the intact protein. The combined results indicate that the interknuckle interactions, which involve the close approach of the side-chains of Phe16 and Trp37, are transitory. The solution behavior of NC may be best considered as a rapid equilibrium between conformations with weakly interacting and non-interacting knuckle domains. This inherent conformational flexibility may be functionally important, enabling adaptive binding of NC to different recognition elements within the HIV-1 psi-RNA packaging signal.


Subject(s)
HIV-1/chemistry , Nucleocapsid Proteins/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry
6.
Science ; 279(5349): 384-8, 1998 Jan 16.
Article in English | MEDLINE | ID: mdl-9430589

ABSTRACT

The three-dimensional structure of the human immunodeficiency virus-type 1 (HIV-1) nucleocapsid protein (NC) bound to the SL3 stem-loop recognition element of the genomic Psi RNA packaging signal has been determined by heteronuclear magnetic resonance spectroscopy. Tight binding (dissociation constant, approximately 100 nM) is mediated by specific interactions between the amino- and carboxyl-terminal CCHC-type zinc knuckles of the NC protein and the G7 and G9 nucleotide bases, respectively, of the G6-G7-A8-G9 RNA tetraloop. A8 packs against the amino-terminal knuckle and forms a hydrogen bond with conserved Arg32, and residues Lys3 to Arg10 of NC form a 310 helix that binds to the major groove of the RNA stem and also packs against the amino-terminal zinc knuckle. The structure provides insights into the mechanism of viral genome recognition, explains extensive amino acid conservation within NC, and serves as a basis for the development of inhibitors designed to interfere with genome encapsidation.


Subject(s)
Gene Products, gag/chemistry , HIV-1/chemistry , Nucleocapsid/chemistry , RNA, Viral/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Gene Products, gag/metabolism , Genome, Viral , HIV-1/genetics , Hydrogen Bonding , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Nucleic Acid Conformation , Nucleocapsid/metabolism , Protein Conformation , Protein Folding , Protein Structure, Secondary , RNA, Viral/genetics , RNA, Viral/metabolism , Zinc/chemistry , Zinc/metabolism , Zinc Fingers
7.
Biopolymers ; 48(2-3): 181-95, 1998.
Article in English | MEDLINE | ID: mdl-10333745

ABSTRACT

The x-ray structure of the glutamine aminoacyl tRNA synthetase bound to its cognate tRNA(Gln) and ATP was reported by Steitz and co-workers in 1989, providing the first high resolution structure of a protein-RNA complex. Since then, high resolution structures have been reported for RNA complexes with five other tRNA synthetases, the elongation factor Tu, the bacteriophage MS2 coat protein, the human spliceosomal U1A and U2B"-U1A' proteins, and the HIV-1 nucleocapsid protein. Although the number of high resolution structures of protein-RNA complexes are rather small, some general themes have begun to emerge regarding the nature and mechanisms of protein-RNA recognition.


Subject(s)
RNA-Binding Proteins/metabolism , Amino Acyl-tRNA Synthetases/metabolism , Humans , Models, Molecular , Nucleic Acid Conformation
8.
J Med Chem ; 40(13): 1969-76, 1997 Jun 20.
Article in English | MEDLINE | ID: mdl-9207937

ABSTRACT

The highly conserved and mutationally intolerant retroviral zinc finger motif of the HIV-1 nucleocapsid protein (NC) is an attractive target for drug therapy due to its participation in multiple stages of the viral replication cycle. A literature search identified cystamine, thiamine disulfide, and disulfiram as compounds that have been shown to inhibit HIV-1 replication by poorly defined mechanisms and that have electrophilic functional groups that might react with the metal-coordinating sulfur atoms of the retroviral zinc fingers and cause zinc ejection. 1H NMR studies reveal that these compounds readily eject zinc from synthetic peptides with sequences corresponding to the HIV-1 NC zinc fingers, as well as from the intact HIV-1 NC protein. In contrast, the reduced forms of disulfiram and cystamine, diethyl dithiocarbamate and cysteamine, respectively, were found to be ineffective at zinc ejection, although cysteamine formed a transient complex with the zinc fingers. Studies with HIV-1-infected human T-cells and monocyte/macrophage cultures revealed that cystamine and cysteamine possess significant antiviral properties at nontoxic concentrations, which warrant their consideration as therapeutically useful anti-HIV agents.


Subject(s)
Acquired Immunodeficiency Syndrome/drug therapy , Capsid Proteins , Capsid/metabolism , Cystamine/therapeutic use , Disulfiram/therapeutic use , Gene Products, gag/metabolism , Thiamine/analogs & derivatives , Viral Proteins , Zinc Fingers , Amino Acid Sequence , Anti-HIV Agents/chemistry , Anti-HIV Agents/therapeutic use , Cells, Cultured , Cystamine/chemistry , Disulfiram/chemistry , Ditiocarb/chemistry , Ditiocarb/pharmacology , Ditiocarb/therapeutic use , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Thiamine/chemistry , Thiamine/therapeutic use , gag Gene Products, Human Immunodeficiency Virus
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