Nucleic acid binding properties of the simian immunodeficiency virus nucleocapsid protein NCp8

The nucleocapsid protein of simian immunodeficiency virus (SIV) NCp8 has two copies of conserved sequences (termed zinc fingers, ZF) of 14 amino acids with 4 invariant residues (CCHC) that coordinate Zn(II). Each of its two ZFs has a Trp residue. A significant quenching of NCp8 Trp fluorescence was seen in nucleic acid complexes, suggesting stacking of the indole ring with nucleobases and the simultaneous involvement of both ZFs in the binding process. Both ZFs contribute to the nucleic acid binding free energy of NCp8, albeit in a not additive manner. NCp8 exhibited a base preference analogous to that of NCp7: G approximately I > T > U > C > A. Alternating base sequences that bind HIV-1 NCp7 in a sequence-specific manner were also bound selectively by NCp8. Specific sequence recognition required at least five bases and the presence of bound Zn(II). The two ZFs account for the net displacement of 3 out of 4 sodium ions upon binding (2 by the first and one by the second finger), and for most (85%) of the hydrophobic stabilization in complex formation. Based on the sequence and functional similarity of SIV NCp8 and HIV-1 NCp7, and using available structural information for free and oligonucleotide bound NCp7, we propose a structural model for NCp8-oligonucleotide complexes.     

Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study.

The steady-state and time-resolved fluorescence properties of two zinc-saturated 18-residue synthetic peptides with the amino acid sequence of the NH2-terminal (NCp7 13-30 F16W, where the naturally occurring Phe was replaced by a Trp residue) and the COOH-terminal (NCp7 34-51) zinc finger domains of human immunodeficiency virus type I nucleocapsid protein were investigated. Fluorescence intensity decay of both Trp 16 and Trp 37 residues suggested the existence of two fully solvent-exposed ground-state classes governed by a C = 2.2 equilibrium constant. The lifetimes of Trp 16 classes differed from those of Trp 37 essentially because of differences in nonradiative rate constants. Arrhenius plots of the temperature-dependent nonradiative rate constants suggested that the fluorescence quenchers involved in both classes and in both peptides were different and the collisional rate of these quenchers with the indole ring was very low, probably because of the highly constrained peptide chain conformation. The nature of the ground-state classes was discussed in relation to 1H nuclear magnetic resonance data. Using Trp fluorescence to monitor the interaction of both peptides with tRNA(Phe) we found that a stacking between the indole ring of both Trp residues and the bases of tRNA(Phe) occurred. This stacking constituted the main driving force of the interaction and modified the tRNA(Phe) conformation. Moreover, the binding of both fingers to tRNA(Phe) was noncooperative with similar site size (3 nucleotide residues/peptide), but the affinity of the NH2-terminal finger domain (K = 1.3 (+/- 0.2) 10(5) M-1) in low ionic strength buffer was one order of magnitude larger than the COOH-terminal one due to additional electrostatic interactions involving Lys 14 and/or Arg 29 residues.     

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.     

Phosphorescence and optically detected magnetic resonance of HIV-1 nucleocapsid protein complexes with stem-loop sequences of the genomic Psi-recognition element

The binding of NCp7, the nucleocapsid protein of human immunodeficiency virus type 1, to oligonucleotide stem--loop (SL) sequences of the genomic Psi-recognition element has been studied using fluorescence, phosphorescence, and optically detected magnetic resonance (ODMR). RNA SL2, SL3, and SL4 constructs bind with higher affinity than the corresponding DNAs. G to I substitutions in the SL3 DNA loop sequence lead to reduced binding affinity and significant changes in the triplet state properties of Trp37 of NCp7, implicating these bases in contacts with aromatic amino acid residues of the zinc finger domains of NCp7, in agreement with the NMR structure of the 1:1 complex of NCp7 and SL3 RNA [DeGuzman, R. N., Wu, Z. R., Stalling, C. C., Pappaladro, L., Borer, P. N., and Summers, M. F. (1998) Science 279, 384-388]. The NCp7 to SL binding stoichiometry is 2:1 for intact SL sequences but is reduced to 1:1 for SL variants with an abasic or hydrocarbon loop. It is proposed that Delta D/Delta E(0,0), where Delta D is the change in the zero-field splitting D parameter and Delta E(0,0) is the shift of the tryptophan phosphorescence origin, provides a measure of aromatic stacking interactions with nucleic acid bases. Values on the order of 10(-5) indicate significant stacking interactions, while values closer to 10(-6) result from interactions not involving aromatic stacking. Binding of NCp7 to oligonucleotide substrates produces shortened Trp37 triplet state lifetimes by enhancement of k(x) and an increase of the relative value of P(x), the intersystem crossing rate to the T(x) sublevel. These effects are attributed to a reduction in the degree of electronic symmetry of Trp37 in the complexes. Guanine and adenine triplet states produced by optical pumping of SL3 DNA are characterized. We find, as with tryptophan, that D < 3E.     

Effect of nucleic acid binding on the triplet state properties of tetrapeptides containing tryptophan and 6-methyltryptophan: a study by phosphorescence and ODMR spectroscopy

Complexes of four peptides [KWGK, KGWK, K(6MeW)GK, KG(6MeW)K] with the nucleic acids [poly(A), poly(C), poly(U), poly(I), and rG(8)] have been investigated by phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy. The intrinsic spectroscopic probes used in these studies are tryptophan (W) and 6-methyltryptophan (6MeW). Binding to the nucleic acids results in a red-shift of the phosphorescence 0,0-band (delta E(0,0)) of the aromatic residue as well as a reduction of its zero-field splitting parameter (delta D). Results are compared with earlier studies of the HIV-1 nucleocapsid protein, NCp7, that contains a single tryptophan residue (Trp37) within a retroviral zinc finger sequence. Binding of poly(A) or poly(U) to the tetrapeptides induces larger delta E(0,0) and delta D than when bound to NCp7, indicating stronger stacking interactions. Poly(I), on the other hand, produces larger shifts in Trp37 of NCp7. Binding of rG(8) produces sequence-dependent effects in the peptides. When bound to NCp7, but in contrast with tetrapeptide binding, nucleic acids produce large changes in triplet state kinetics consistent with enhanced spin-orbit coupling. These results are discussed in terms of three limiting types of tryptophan-base interaction: intercalation, aromatic stacking, and edge-on interaction. These should have differing effects on the properties of the triplet state.     

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.     

Zn(2+) binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein: evidence of a critical role of cysteine 49 in Zn(2+) dissociation

The two highly conserved Zn(2+) finger motifs of the HIV-1 nucleocapsid protein, NCp7, strongly bind Zn(2+) through coordination of one His and three Cys residues. To further analyze the role of these residues, we investigated the Zn(2+) binding and acid-base properties of four single-point mutants of a short peptide corresponding to the distal finger motif of NCp7. In each mutant, one Zn(2+)-coordinating residue is substituted with a noncoordinating one. Using the spectroscopic properties of Co(2+), we first establish that the four mutants retain their ability to bind a metal cation through a four- or five-coordinate geometry with the vacant ligand position(s) presumably occupied by water molecule(s). Moreover, the pK(a) values of the three Cys residues of the mutant apopeptide where His44 is substituted with Ala are found by (1)H NMR to be similar to those of the native peptide, suggesting that the mutations do not affect the acid-base properties of the Zn(2+)-coordinating residues. The binding of Zn(2+) was monitored by using the fluorescence of Trp37 as an intrinsic probe. At pH 7.5, the apparent Zn(2+) binding constants (between 1.6 x 10(8) and 1.3 x 10(10) M(-)(1)) of the four mutants are strongly reduced compared to those of the native peptide but are similar to those of various host Zn(2+) binding proteins. As a consequence, the loss of viral infectivity following the mutation of one Zn(2+)-coordinating residue in vivo may not be related to the total loss of Zn(2+) binding. The pH dependence of Zn(2+) binding indicates that the coordinating residues bind Zn(2+) stepwise and that the free energy provided by the binding of a given residue may be modulated by the entropic contribution of the residues already bound to Zn(2+). Finally, the pK(a) of Cys49 in the holopeptide is found to be 5.0, a value that is at least 0.7 unit higher than those for the other Zn(2+)-coordinating residues. This implies that Cys49 may act as a switch for Zn(2+) dissociation in the distal finger motif of NCp7, a feature that may contribute to the high susceptibility of Cys49 to electrophilic attack.     

Effects of temperature on the dynamic behaviour of the HIV-1 nucleocapsid NCp7 and its DNA complex

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) contains two highly conserved CCHC zinc fingers and is involved in many crucial steps of the virus life-cycle. A large number of physiological r?les of NCp7 involve its binding to single-stranded nucleic acid chains. Several solution structures of NCp7 and its complex with single-stranded RNA or DNA have been reported. We have investigated the changes in the dynamic behaviour experienced by the (12-53)NCp7 peptide upon DNA binding using (15)N heteronuclear relaxation measurements at 293 K and 308 K, and fluorescence spectroscopy. The relaxation data were interpreted using the reduced spectral density approach, which allowed the high-frequency motion, overall tumbling rates and the conformational exchange contributions to be characterized for various states of the peptide without using a specific motional model. Analysis of the temperature-dependent correlation times derived from both NMR and fluorescence data indicated a co-operative change of the molecular shape of apo (12-53)NCp7 around 303 K, leading to an increased hydrodynamic radius at higher temperatures. The binding of (12-53)NCp7 to a single-stranded d(ACGCC) pentanucleotide DNA led to a reduction of the conformational flexibility that characterized the apo peptide. Translational diffusion experiments as well as rotational correlation times indicated that the (12-53)NCp7/d(ACGCC) complex tumbles as a rigid object. The amplitudes of high-frequency motions were restrained in the complex and the occurrence of conformational exchange was displaced from the second zinc finger to the linker residue Ala30.     

Molecular mechanism of the Zn2+-induced folding of the distal CCHC finger motif of the HIV-1 nucleocapsid protein

HIV-1 nucleocapsid protein, NCp7, contains two highly conserved CCHC zinc fingers. Binding of Zn(2+) drives NCp7 from an unfolded to a highly folded structure that is critical for its functions. Using the intrinsic fluorescence of Trp(37), we investigated, by the stopped-flow technique, the folding of NCp7 distal finger through the pH dependence of its Zn(2+) association and dissociation kinetics. Zn(2+) binding was found to involve four different paths associated with the four deprotonated states of the finger. Each binding path involves the rapid formation of an intermediate complex that is subsequently rearranged and stabilized in a rate-limiting step. The equilibrium and kinetic rate constants of the full Zn(2+)-binding process have been determined. At neutral pH, the preferential pathway for the Zn(2+)-driven folding implies Zn(2+) binding to the deprotonated Cys(36) and His(44) residues, in the bidentate state of the finger. The resulting intermediate is then converted with a rate constant of 500 s(-1) into a more suitably folded form, probably through a rearrangement of the peptide backbone around Zn(2+) to optimize the binding geometry. This form then rapidly leads to the final native complex, through deprotonation of Cys(39) and Cys(49) residues and intramolecular substitution of coordinated water molecules. Zn(2+) dissociation is also characterized by a multistep process and occurs fastest via the deprotonated Zn(2+)-bound bidentate state with a rate constant of 3 s(-1). Due to their critical role in folding, the intermediates identified for the first time in this study may constitute potential targets for HIV therapy.     

Physical studies of tyrosine and tryptophan residues in mammalian A1 heterogeneous nuclear ribonucleoprotein. Support for a segmented structure.

The mammalian heterogeneous ribonucleoprotein (hnRNP) A1 and its constituent N-terminal domain, termed UP1, have been studied by steady-state and dynamic fluorimetry, as well as phosphorescence and optically detected magnetic resonance (ODMR) spectroscopy at cryogenic temperatures. The results of these diverse techniques coincide in assigning the site of the single tryptophan residue of A1, located in the UP1 domain, to a partially solvent-exposed site distal to the protein's nucleic acid binding surface. In contrast, tyrosine fluorescence is significantly perturbed when either protein associates with single-stranded polynucleotides. Tyr to Trp energy transfer at the singlet level is found for both UP1 and A1 proteins. Single-stranded polynucleotide binding induces a quenching of their intrinsic fluorescence emission, which can be attributed to a significant reduction (greater than 50%) of the Tyr contribution, while Trp emission is only quenched by approximately 15%. Tyrosine quenching effects of similar magnitude are seen upon polynucleotide binding by either UP1 (1 Trp, 4 Tyr) or A1 (1 Trp, 12 Tyr), strongly suggesting that Tyr residues in both the N-terminal and C-terminal domain of A1 are involved in the binding process. Tyr phosphorescence emission was strongly quenched in the complexes of UP1 with various polynucleotides, and was attributed to triplet state energy transfer to nucleic acid bases located in the close vicinity of the fluorophore. These results are consistent with stacking of the tyrosine residues with the nucleic acid bases. While the UP1 Tyr phosphorescence lifetime is drastically shortened in the polynucleotide complex, no change of phosphorescence emission maximum, phosphorescence decay lifetime or ODMR transition frequencies were observed for the single Trp residue. The results of dynamic anisotropy measurements of the Trp fluorescence have been interpreted as indicative of significant internal flexibility in both UP1 and A1, suggesting a flexible linkage connecting the two sub-domains in UP1. Theoretical calculations based on amino acid sequence for chain flexibility and other secondary structural parameters are consistent with this observation, and suggest that flexible linkages between sub-domains may exist in other RNA binding proteins. While the dynamic anisotropy data are consistent with simultaneous binding of both the C-terminal and the N-terminal domains to the nucleic acid lattice, no evidence for simultaneous binding of both UP1 sub-domains was found.