Targeting of murine leukemia virus gag to the plasma membrane is mediated by PI(4,5)P2/PS and a polybasic region in the matrix.

Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P(2) only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P(2) depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P(2) appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P(2) binding cleft.

Azodicarbonamide inhibits HIV-1 replication by targeting the nucleocapsid protein.

Nucleocapsid p7 (NCp7) proteins of human immunodeficiency virus type 1 (HIV-1) contain two zinc binding domains of the sequence Cys-(X)2-Cys-(X)4-His-(X)4-Cys (CCHC). The spacing pattern and metal-chelating residues (3 Cys, 1 His) of these nucleocapside CCHC zinc fingers are highly conserved among retroviruses. These CCHC domains are required during both the early and late phases of retroviral replication, making them attractive targets for antiviral agents. toward that end, we have identified a number of antiviral chemotypes that electrophilically attack the sulfur atoms of the zinc-coordinating cysteine residues of the domains. Such nucleocapside inhibitors were directly virucidal by preventing the initiation of reverse transcription and blocked formation of infectious virus from cells through modification of CCHC domains within Gag precursors. Herein we report that azodicarbonamide (ADA) represents a new compound that inhibits HIV-1 and a broad range of retroviruses by targeting the the nucleocapsid CCHC domains. Vandevelde et al. also recently disclosed that ADA inhibits HIV-1 infection via an unidentified mechanism and that ADA was introduced into Phase I/II clinical trials in Europe for advanced AIDS. These studies distinguish ADA as the first known nucleocapsid inhibitor to progress to human trials and provide a lead compound for drug optimization.

Structure of the HIV-1 nucleocapsid protein bound to the SL3 psi-RNA recognition element.

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.

Structure of the amino-terminal core domain of the HIV-1 capsid protein.

The three-dimensional structure of the amino-terminal core domain (residues 1 through 151) of the human immunodeficiency virus-type 1 (HIV-1) capsid protein has been solved by multidimensional heteronuclear magnetic resonance spectroscopy. The structure is unlike those of previously characterized viral coat proteins and is composed of seven alpha helices, two beta hairpins, and an exposed partially ordered loop. The domain is shaped like an arrowhead, with the beta hairpins and loop exposed at the trailing edge and the carboxyl-terminal helix projecting from the tip. The proline residue Pro1 forms a salt bridge with a conserved, buried aspartate residue (Asp51), which suggests that the amino terminus of the protein rearranges upon proteolytic maturation. The binding site for cyclophilin A, a cellular rotamase that is packaged into the HIV-1 virion, is located on the exposed loop and encompasses the essential proline residue Pro90. In the free monomeric domain, Pro90 adopts kinetically trapped cis and trans conformations, raising the possibility that cyclophilin A catalyzes interconversion of the cis- and trans-Pro90 loop structures.

Solution structure of 3-oxo-delta5-steroid isomerase.

The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

Structural biology of HIV.

The human immunodeficiency virus (HIV) genome encodes a total of three structural proteins, two envelope proteins, three enzymes, and six accessory proteins. Studies over the past ten years have provided high-resolution three-dimensional structural information for all of the viral enzymes, structural proteins and envelope proteins, as well as for three of the accessory proteins. In some cases it has been possible to solve the structures of the intact, native proteins, but in most cases structural data were obtained for isolated protein domains, peptidic fragments, or mutants. Peptide complexes with two regulatory RNA fragments and a protein complex with an RNA recognition/encapsidation element have also been structurally characterized. This article summarizes the high-resolution structural information that is currently available for HIV proteins and reviews current structure-function and structure-biological relationships.

Solution structure of the capsid protein from the human T-cell leukemia virus type-I.

The solution structure of the capsid protein (CA) from the human T-cell leukemia virus type one (HTLV-I), a retrovirus that causes T-cell leukemia and HTLV-I-associated myelopathy in humans, has been determined by NMR methods. The protein consists of independent N and C-terminal domains connected by a flexible linker. The domains are structurally similar to the N-terminal "core" and C-terminal "dimerization" domains, respectively, of the human immunodeficiency virus type one (HIV-1) and equine infectious anemia virus (EIAV) capsid proteins, although several important differences exist. In particular, hydrophobic residues near the major homology region are partially buried in HTLV-I CA, which is monomeric in solution, whereas analogous residues in HIV-1 and EIAV CA project from the C-terminal domain and promote dimerization. These differences in the structure and oligomerization state of the proteins appear to be related to, and possibly controlled by, the oxidation state of conserved cysteine residues, which are reduced in HTLV-I CA but form a disulfide bond in the HIV-1 and EIAV CA crystal structures. The results are consistent with an oxidative capsid assembly mechanism, in which CA oligomerization or maturation is triggered by disulfide bo nd formation as the budding virus enters the oxidizing environment of the bloodstream.

NMR structure of HMfB from the hyperthermophile, Methanothermus fervidus, confirms that this archaeal protein is a histone.

The three-dimensional structure of the recombinant histone rHMfB from Methanothermus fervidus, an archaeon that grows optimally at 83 degrees C, has been determined by nuclear magnetic resonance methods. This is only the third structure of a protein from a hyperthermophilic organism (optimal growth at temperatures above 80 degrees C). Signal assignments were made using a combination of homonuclear-correlated, 15N-double resonance and 15N, 13C triple resonance NMR experiments. Long range dipolar interactions for the symmetric homodimer were identified from two-dimensional 13C-double half-filtered and three-dimensional 13C-filtered NMR data obtained for a heterolabeled-dimer. A family of 33 structures was calculated using DSPACE with a total of 609 NOE-derived interproton distance restraints, including 22 intraresidue, 192 sequential, 300 medium-range (two to five residues), 86 long-range intramolecular (more than five residues) and 112 intermolecular distance restraints. The monomer subunits consist of three alpha-helices, extending from residues Pro4 to Ala15 (helix I), Ser21 to Ala50 (helix II) and Lys56 to Lys68 (helix III), as well as two short segments of beta-strand comprised of residues Arg19 to Ser21 and Thr54 to Ile55. Helices I, II and III contain N-terminal capping boxes, and helices I and II contain C-terminal caps. The structure of the (rHMfB)2 dimer appears very similar to the dimer subunits within the histone core octamer of the chicken nucleosome. The presence of a canonical "histone fold" motif in rHMfB is consistent with the HMf family of archaeal histones and the eukaryal nucleosome core histones having evolved from a common ancestor. The (rHMfB)2 dimer contains several structural features that may impart thermal stability (or non-lability), including two novel hydrophobic "proline Ncaps", four interhelical hydrogen bonds and short N- and C-terminal disordered tails.

NMR studies of the pbx1 TALE homeodomain protein free in solution and bound to DNA: proposal for a mechanism of HoxB1-Pbx1-DNA complex assembly.

The Hox homeodomain proteins are transcription factors involved in developmental regulation. Many of the vertebrate Hox proteins bind DNA cooperatively with the Pbx1 homeodomain protein. The crystal structure of a human HoxB1-Pbx1-DNA ternary complex revealed that interactions between the two proteins are mediated by the HoxB1 hexapeptide, which inserts into a hydrophobic pocket in Pbx1. It was also found that the Pbx1 DNA-binding domain is larger than the canonical three-helix homeodomain, containing an additional alpha-helix that is joined to the C terminus of the homeodomain by a turn of 310helix. These extra C-terminal residues had previously been shown to augment the cooperative interaction of Pbx1 with Hox partners, as well as enhancing the DNA binding of monomeric Pbx1. In order to characterize the role of the fourth Pbx1 helix in greater detail, we have examined the backbone structure of the enlarged Pbx1 DNA-binding domain in solution by(1)H,(15)N and(13)C multidimensional NMR spectroscopy. Our results show that the additional alpha-helix of Pbx1 is unfolded when the protein is free in solution and that its folding is triggered by binding of Pbx1 to DNA. In contrast, no change in conformation is observed upon mixing the HoxB1 protein with Pbx1 in the absence of DNA. This study suggests a model for the assembly of a stable HoxB1-Pbx1-DNA ternary complex.

Solution structure and dynamics of the Rous sarcoma virus capsid protein and comparison with capsid proteins of other retroviruses.

The solution structure and dynamics of the recombinant 240 amino acid residue capsid protein from the Rous sarcoma virus has been determined by NMR methods. The structure was determined using 2200 distance restraints and 330 torsion angle restraints, and the dynamics analysis was based on (15)N relaxation parameters (R(1), R(2), and (1)H-(15)N NOE) measured for 153 backbone amide groups. The monomeric protein consists of independently folded N- and C-terminal domains that comprise residues Leu14-Leu146 and Ala150-Gln226, respectively. The domains exhibit different rotational correlation times (16.6(+/-0.1) ns and 12.6(+/-0.1) ns, respectively), are connected by a flexible linker (Ala147-Pro149), and do not give rise to inter-domain NOE values, indicating that they are dynamically independent. Despite limited sequence similarity, the structure of the Rous sarcoma virus capsid protein is similar to the structures determined recently for the capsid proteins of retroviruses belonging to the lentivirus and human T-cell leukemia virus/bovine leukemia virus genera. Structural differences that exist in the C-terminal domain of Rous sarcoma virus capsid relative to the other capsid proteins appear to be related to the occurrence of conserved cysteine residues. Whereas most genera of retroviruses contain a pair of conserved and essential cysteine residues in the C-terminal domain that appear to function by forming an intramolecular disulfide bond during assembly, the Rous sarcoma virus capsid protein does not. Instead, the Rous sarcoma virus capsid protein contains a single cysteine residue that appears to be conserved among the avian C-type retroviruses and is positioned in a manner that might allow the formation of an intermolecular disulfide bond during capsid assembly.

Three-dimensional structure of the human immunodeficiency virus type 1 matrix protein.

The HIV-1 matrix protein forms an icosahedral shell associated with the inner membrane of the mature virus. Genetic analyses have indicated that the protein performs important functions throughout the viral life-cycle, including anchoring the transmembrane envelope protein on the surface of the virus, assisting in viral penetration, transporting the proviral integration complex across the nuclear envelope, and localizing the assembling virion to the cell membrane. We now report the three-dimensional structure of recombinant HIV-1 matrix protein, determined at high resolution by nuclear magnetic resonance (NMR) methods. The HIV-1 matrix protein is the first retroviral matrix protein to be characterized structurally and only the fourth HIV-1 protein of known structure. NMR signal assignments required recently developed triple-resonance (1H, 13C, 15N) NMR methodologies because signals for 91% of 132 assigned H alpha protons and 74% of the 129 assignable backbone amide protons resonate within chemical shift ranges of 0.8 p.p.m. and 1 p.p.m., respectively. A total of 636 nuclear Overhauser effect-derived distance restraints were employed for distance geometry-based structure calculations, affording an average of 13.0 NMR-derived distance restraints per residue for the experimentally constrained amino acids. An ensemble of 25 refined distance geometry structures with penalties (sum of the squares of the distance violations) of 0.32 A2 or less and individual distance violations under 0.06 A was generated; best-fit superposition of ordered backbone heavy atoms relative to mean atom positions afforded root-mean-square deviations of 0.50 (+/- 0.08) A. The folded HIV-1 matrix protein structure is composed of five alpha-helices, a short 3(10) helical stretch, and a three-strand mixed beta-sheet. Helices I to III and the 3(10) helix pack about a central helix (IV) to form a compact globular domain that is capped by the beta-sheet. The C-terminal helix (helix V) projects away from the beta-sheet to expose carboxyl-terminal residues essential for early steps in the HIV-1 infectious cycle. Basic residues implicated in membrane binding and nuclear localization functions cluster about an extruded cationic loop that connects beta-strands 1 and 2. The structure suggests that both membrane binding and nuclear localization may be mediated by complex tertiary structures rather than simple linear determinants.

Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Psi-RNA packaging signal: implications for genome recognition.

Murine leukemia virus (MLV) is currently the most widely used gene delivery system in gene therapy trials. The simple retrovirus packages two copies of its RNA genome by a mechanism that involves interactions between the nucleocapsid (NC) domain of a virally-encoded Gag polyprotein and a segment of the RNA genome located just upstream of the Gag initiation codon, known as the Psi-site. Previous studies indicated that the MLV Psi-site contains three stem loops (SLB-SLD), and that stem loops SLC and SLD play prominent roles in packaging. We have developed a method for the preparation and purification of large quantities of recombinant Moloney MLV NC protein, and have studied its interactions with a series of oligoribonucleotides that contain one or more of the Psi-RNA stem loops. At RNA concentrations above approximately 0.3 mM, isolated stem loop SLB forms a duplex and stem loops SL-C and SL-D form kissing complexes, as expected from previous studies. However, neither the monomeric nor the dimeric forms of these isolated stem loops binds NC with significant affinity. Longer constructs containing two stem loops (SL-BC and SL-CD) also exhibit low affinities for NC. However, NC binds with high affinity and stoichiometrically to both the monomeric and dimeric forms of an RNA construct that contains all three stem loops (SL-BCD; K(d)=132(+/-55) nM). Titration of SL-BCD with NC also shifts monomer-dimer equilibrium toward the dimer. Mutagenesis experiments demonstrate that the conserved GACG tetraloops of stem loops C and D do not influence the monomer-dimer equilibrium of SL-BCD, that the tetraloop of stem loop B does not participate directly in NC binding, and that the tetraloops of stem loops C and D probably also do not bind to NC. These surprising results differ considerably from those observed for HIV-1, where NC binds to individual stem loops with high affinity via interactions with exposed residues of the tetraloops. The present results indicate that MLV NC binds to a pocket or surface that only exists in the presence of all three stem loops.

Dynamical behavior of the HIV-1 nucleocapsid protein.

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.

Three-dimensional structure of the HTLV-II matrix protein and comparative analysis of matrix proteins from the different classes of pathogenic human retroviruses.

The matrix protein performs similar roles in all retroviruses, initially directing membrane localization of the assembling viral particle and subsequently forming a stable structural shell associated with the inner surface of the mature viral membrane. Although conserved structural elements are likely to perform these functions in all retroviral matrix proteins, invariant motifs are not evident at the primary sequence level and three-dimensional structures have been available for only the primate lentiviral matrix proteins. We have therefore used NMR spectroscopy to determine the structure of the matrix protein from human T-cell leukemia virus type II (HTLV-II), a member of the human oncovirus subclass of retroviruses. A total of 577 distance restraints were used to build 20 refined models that superimpose with an rmsd of 0.71 A for the backbone atoms of the structured regions. The globular HTLV-II matrix structure is composed of four alpha-helices and a 3(10) helix. Exposed basic residues near the C terminus of helix II form a putative membrane binding surface which could act in concert with the N-terminal myristoyl group to anchor the protein on the viral membrane surface. Clear structural similarities between the HTLV-II and HIV-1 matrix proteins suggest that the topology and exposed cationic membrane binding surface are likely to be conserved features of retroviral matrix proteins.

NMR structure of stem-loop SL2 of the HIV-1 psi RNA packaging signal reveals a novel A-U-A base-triple platform.

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.

Stem-loop SL4 of the HIV-1 psi RNA packaging signal exhibits weak affinity for the nucleocapsid protein. structural studies and implications for genome recognition.

Encapsidation of the genome of the human immunodeficiency virus type-1 (HIV-1) during retrovirus assembly is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and a approximately 110 nucleotide segment of the genome known as the Psi-site. The HIV-1 Psi-site contains four stem-loops (SL1 through SL4), all of which are important for genome packaging. Recent isothermal titration calorimetry (ITC) studies have demonstrated that SL2 and SL3 are capable of binding NC with high affinity (K(d) approximately 140 nM), consistent with proposals for protein-interactive functions during packaging. To determine if SL4 may have a similar function, NC-interactive studies were conducted by NMR and gel-shift methods. In contrast to previous reports, we find that SL4 binds weakly to NC (K(d)=(+/-14 microM), suggesting an alternative function. NMR studies indicate that the GAGA tetraloop of SL4 adopts a classical GNRA-type fold (R=purine, N=G, C, A or U), a motif that stabilizes RNA tertiary structures in other systems. In combination with previously reported gel mobility studies of Psi-site deletion mutants, these findings suggest that SL4 functions in genome recognition not by binding to Gag, but by stabilizing the structure of the Psi-site. Differences in the affinities of NC for SL2, SL3 and SL4 stem-loops can now be rationalized in terms of the different structural properties of stem loops that contain GGNG (SL2 and SL3) and GNRA (SL4) sequences.

Structure of the cysteine-rich intestinal protein, CRIP.

LIM domains are Zn-binding arrays found in a number of proteins involved in the control of cell differentiation, including several developmentally regulated transcription factors and a human proto-oncogene product. The rat cysteine-rich intestinal protein, CRIP, is a 76-residue polypeptide which contains a LIM motif. The solution structure of CRIP has been determined by homonuclear and 1H-15N heteronuclear correlated nuclear magnetic resonance spectroscopy. Structures with individual distance violations of < or = 0.03 angstrom and penalties (squared sum of distance violations) of < or = 0.06 angstrom2 were generated with a total of 500 nuclear Overhauser effect (NOE)-derived distance restraints (averaging 15.6 restraints per refined residue). Superposition of backbone heavy atoms of ordered residues relative to mean atom positions is achieved with pairwise rms deviations of 0.54(+/-0.14) angstrom. As observed previously for a peptide with the sequence of the C-terminal LIM domain from the avian cysteine-rich protein, CRP (cCRP-LIM2), CRIP binds two equivalents of zinc, forming N-terminal CCHC (Cys3, Cys6, His24, Cys27) and C-terminal CCCC (Cys30, Cys33, Cys51, Cys55) modules. The CCHC and CCCC modules in CRIP contain two orthogonally-arrayed antiparallel beta-sheets. The C-terminal end of the CCHC module contains a tight turn and the C terminus of the CCCC module forms an alpha-helix. The modules pack via hydrophobic interactions, forming a compact structure that is similar to that observed for cCRP-LIM2. The most significant differences between the structures occur at the CCHC module-CCCC module interface, which results in a difference in the relative orientations of the modules, and at the C terminus where the alpha-helix appears to be packed more tightly against the preceding antiparallel beta-sheet. The greater abundance of NOE information obtained for CRIP relative to cCRP-LIM2, combined with the analysis of J-coupling and proton chemical shift data, have allowed a more detailed evaluation of the molecular level interactions that stabilize the fold of the LIM motif.

NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition.

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.

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.

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.