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.

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.

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.

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.

A mechanism for plus-strand transfer enhancement by the HIV-1 nucleocapsid protein during reverse transcription.

The HIV-1 nucleocapsid protein (NC) functions as a nucleic acid chaperone during the plus-strand transfer step in reverse transcription by facilitating annealing of the primer binding site (PBS) sequence in the short plus-strand strong-stop DNA fragment [(+) SSDNA] to a complementary site located near the 3' end of the minus-strand DNA [(-) PBS DNA]. To investigate the mechanism by which NC performs this function, we have prepared an 18-nucleotide (-) PBS DNA for nuclear magnetic resonance (NMR) based structural and NC binding studies. The (-) PBS DNA forms a stable hairpin (T(m) approximately 42 +/- 5 degrees C) that contains a five-residue loop and a bulged thymine in a guanosine-cytosine-rich stem. Addition of substoichiometric amounts of NC results in significant broadening and reductions in NMR signal intensities of the Watson-Crick base-paired imino protons and a reduction by 20 degrees C in the upper temperature at which the imino proton signals are detectable, consistent with destabilization of the structure. The results suggest that inefficient annealing in the absence of NC may be due to the intrinsic stability of an internal (-) PBS DNA hairpin and that NC facilitates strand transfer by destabilizing the hairpin and exposing stem nucleotides for base pairing with the PBS sequence in (+) SSDNA.

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.

The NMR structure of the nucleocapsid protein from the mouse mammary tumor virus reveals unusual folding of the C-terminal zinc knuckle.

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.

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.

Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase.

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through the formation of an intermediate dienolate. The three-dimensional structure of the enzyme from Pseudomonas testosteroni was solved by multidimensional heteronuclear magnetic resonance spectroscopy. This protein, a 28-kDa symmetric dimer, exhibits a three-dimensional fold with the two independently folded monomers packed together via extensive hydrophobic and electrostatic interactions. The previously identified catalytically important residues Tyr-14 (general acid) and Asp-38 (general base) are located near the bottom of a deep hydrophobic cavity and are positioned in a manner consistent with previous mechanistic hypotheses. The structure also revealed the presence of an unexpected acid group (Asp-99) located in the active site adjacent to Tyr-14. Mutagenesis and kinetic studies show that Asp-99 has an anomalously high pK(a) (>9), which allows it to contribute to catalysis by donating a hydrogen bond to the intermediate and to the transition states. In support of this hypothesis, effects on the kinetic parameters of the mutations Y14F and D99A are additive in the Y14F/D99A mutant.

Backbone dynamics of the N-terminal domain of the HIV-1 capsid protein and comparison with the G94D mutant conferring cyclosporin resistance/dependence.

Nuclear magnetic resonance (NMR) (15)N relaxation methods have been used to characterize the backbone dynamics of the N-terminal core domain of the HIV-1 capsid protein (CA(151)). The domain, which has an unusually flat, triangular shape, tumbles in solution at 28 degrees C with an effective rotational correlation time of 11.5 ns. Relaxation data for backbone amides in the domain's seven alpha-helices are indicative of fully anisotropic rotational diffusion. The principal axes of the rotational diffusion tensor calculated from the NMR data are aligned to within 12-23 degrees of the principal axes of the inertial tensor, with the axis of fastest rotational diffusion coincident with that of minimal inertia, and vice versa. Large variations in the (15)N-(1)H nuclear Overhauser effects for individual amino acids correlate with the degree of convergence in the previously calculated NMR structure. In particular, the partially disordered residues Val86-Arg97 that contain the human cyclophilin A (CypA) packaging signal have (15)N heteronuclear NOEs and transversal relaxation rates consistent with a high degree of dynamic conformational averaging. The N-terminal domain of a CA mutant (G94D) that confers both resistance to and dependence on cyclosporin A analogues was also analyzed. Our results indicate that this mutation does not influence the conformation or dynamics of CA(151), and therefore probably affects the function of the protein by modifying essential intermolecular CA-CA interactions.

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 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 of the chicken cysteine-rich protein, CRP1, a double-LIM protein implicated in muscle differentiation.

The mechanism by which the contractile machinery of muscle is assembled and maintained is not well-understood. Members of the cysteine-rich protein (CRP) family have been implicated in these processes. Three vertebrate CRPs (CRP1-3) that exhibit developmentally regulated muscle-specific expression have been identified. All three proteins are associated with the actin cytoskeleton, and one has been shown to be required for striated muscle structure and function. The vertebrate CRPs identified to date display a similar molecular architecture; each protein is comprised of two tandemly arrayed LIM domains, protein-binding motifs found in a number of proteins with roles in cell differentiation. Each LIM domain coordinates two Zn(II) ions that are bound independently in CCHC (C=Cys, H=His) and CCCC modules. Here we describe the solution structure of chicken CRP1 determined by homonuclear and 1H-15N heteronuclear magnetic resonance spectroscopy. Comparison of the structures of the two LIM domains of CRP1 reveals a high degree of similarity in their tertiary folds. In addition, the two component LIM domains represent two completely independent folding units and exhibit no apparent interactions with each other. The structural independence and spatial separation of the two LIM domains of CRP1 are compatible with an adapter or linker role for the protein.

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.

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.

NMR structure and comparison of the archaeal histone HFoB from the mesophile Methanobacterium formicicum with HMfB from the hyperthermophile Methanothermus fervidus.

The solution-state structure of the recombinant archaeal histone rHFoB, from the mesophile Methanobacterium formicicum, has been determined by two- and three-dimensional (3D) proton homonuclear correlated nuclear magnetic resonance (NMR) methods. On the basis of 951 nuclear Overhauser effect (NOE)-derived distance restraints, rHFoB monomers form the histone fold and assemble into symmetric (rHFoB)2 dimers that have a structure consistent with assembly into archaeal nucleosomes. rHFoB exhibits approximately 78% sequence homology with rHMfB from the hyperthermophile Methanothermus fervidus, and the results obtained demonstrate that these two proteins have very similar 3D structures, with a root-mean-square deviation for backbone atoms of 0.65 +/- 0.13 A2. (rHFoB)2 dimers however unfold at lower temperatures and require a higher salt environment for stability than (rHMfB)2 dimers, and comparing the structures, we predict that these differences result from unfavorable surface-located ionic interactions and a larger, more solvent-accessible cavity adjacent to residue G36 in the hydrophobic core of (rHFoB)2.

Solution structure of a zinc domain conserved in yeast copper-regulated transcription factors.

The three dimensional structure of the N-terminal domain (residues 1-42) of the copper-responsive transcription factor Amtl from Candida glabrata has been determined by two-dimensional 1H-correlated nuclear magnetic resonance (NMR) methods. The domain contains an array of zinc-binding residues (Cys-X2-Cys-X8-Cys-X-His) that is conserved among a family of Cu-responsive transcription factors. The structure is unlike those of previously characterized zinc finger motifs, and consists of a three-stranded antiparallel beta-sheet with two short helical segments that project from one end of the beta-sheet. Conserved residues at positions 16, 18 and 19 form a basic patch that may be important for DNA binding.

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.