Conversion of S-phenylsulfonylcysteine residues to mixed disulfides at pH 4.0: utility in protein thiol blocking and in protein-S-nitrosothiol detection.

A three step protocol for protein S-nitrosothiol conversion to fluorescent mixed disulfides with purified proteins, referred to as the thiosulfonate switch, is explored which involves: (1) thiol blocking at pH 4.0 using S-phenylsulfonylcysteine (SPSC); (2) trapping of protein S-nitrosothiols as their S-phenylsulfonylcysteines employing sodium benzenesulfinate; and (3) tagging the protein thiosulfonate with a fluorescent rhodamine based probe bearing a reactive thiol (Rhod-SH), which forms a mixed disulfide between the probe and the formerly S-nitrosated cysteine residue. S-Nitrosated bovine serum albumin and the S-nitrosated C-terminally truncated form of AdhR-SH (alcohol dehydrogenase regulator) designated as AdhR*-SNO were selectively labelled by the thiosulfonate switch both individually and in protein mixtures containing free thiols. This protocol features the facile reaction of thiols with S-phenylsulfonylcysteines forming mixed disulfides at mild acidic pH (pH = 4.0) in both the initial blocking step as well as in the conversion of protein-S-sulfonylcysteines to form stable fluorescent disulfides. Labelling was monitored by TOF-MS and gel electrophoresis. Proteolysis and peptide analysis of the resulting digest identified the cysteine residues containing mixed disulfides bearing the fluorescent probe, Rhod-SH.

Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot.

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.

Equilibrium unfolding pathway of an H-type RNA pseudoknot which promotes programmed -1 ribosomal frameshifting.

The equilibrium unfolding pathway of a 41-nucleotide frameshifting RNA pseudoknot from the gag-pro junction of mouse intracisternal A-type particles (mIAP), an endogenous retrovirus, has been determined through analysis of dual optical wavelength, equilibrium thermal melting profiles and differential scanning calorimetry. The mIAP pseudoknot is an H-type pseudoknot proposed to have structural features in common with the gag-pro frameshifting pseudoknots from simian retrovirus-1 (SRV-1) and mouse mammary tumor virus (MMTV). In particular, the mIAP pseudoknot is proposed to contain an unpaired adenosine base at the junction of the two helical stems (A15), as well as one in the middle of stem 2 (A35). A mutational analysis of stem 1 hairpins and compensatory base-pair substitutions incorporated into helical stem 2 was used to assign optical melting transitions to molecular unfolding events. The optical melting profile of the wild-type RNA is most simply described by four sequential two-state unfolding transitions. Stem 2 melts first in two closely coupled low-enthalpy transitions at low tmin which the stem 3' to A35, unfolds first, followed by unfolding of the remainder of the helical stem. The third unfolding transition is associated with some type of stacking interactions in the stem 1 hairpin loop not present in the pseudoknot. The fourth transition is assigned to unfolding of stem 1. In all RNAs investigated, DeltaHvH approximately DeltaHcal, suggesting that DeltaCpfor unfolding is small. A35 has the thermodynamic properties expected for an extrahelical, unpaired nucleotide. Deletion of A15 destabilizes the stem 2 unfolding transition in the context of both the wild-type and DeltaA35 mutant RNAs only slightly, by DeltaDeltaG degrees approximately 1 kcal mol-1(at 37 degrees C). The DeltaA15 RNA is considerably more susceptible to thermal denaturation in the presence of moderate urea concentrations than is the wild-type RNA, further evidence of a detectable global destabilization of the molecule. Interestingly, substitution of the nine loop 2 nucleotides with uridine residues induces a more pronounced destabilization of the molecule (DeltaDeltaG degrees approximately 2.0 kcal mol-1), a long-range, non-nearest neighbor effect. These findings provide the thermodynamic basis with which to further refine the relationship between efficient ribosomal frameshifting and pseudoknot structure and stability.

Structure, stability and function of RNA pseudoknots involved in stimulating ribosomal frameshifting.

Programmed -1 ribosomal frameshifting has become the subject of increasing interest over the last several years, due in part to the ubiquitous nature of this translational recoding mechanism in pathogenic animal and plant viruses. All cis-acting frameshift signals encoded in mRNAs are minimally composed of two functional elements: a heptanucleotide "slippery sequence" conforming to the general form X XXY YYZ, followed by an RNA structural element, usually an H-type RNA pseudoknot, positioned an optimal number of nucleotides (5 to 9) downstream. The slippery sequence itself promotes a low level ( approximately 1 %) of frameshifting; however, downstream pseudoknots stimulate this process significantly, in some cases up to 30 to 50 %. Although the precise molecular mechanism of stimulation of frameshifting remains poorly understood, significant advances have been made in our knowledge of the three-dimensional structures, thermodynamics of folding, and functional determinants of stimulatory RNA pseudoknots derived from the study of several well-characterized frameshift signals. These studies are summarized here and provide new insights into the structural requirements and mechanism of programmed -1 ribosomal frameshifting.

Base-pairings within the RNA pseudoknot associated with the simian retrovirus-1 gag-pro frameshift site.

Frameshift and readthrough sites within retroviral messenger RNAs are often followed by nucleotide sequences that have the potential to form pseudoknot structures. In the work presented here, NMR methods were used to characterize the base-pairings and structural features of the RNA pseudoknot downstream of the gag-pro frameshift site of simian retrovirus type-1 (SRV-1) and a functional mutant of the SRV-1 pseudoknot. Evidence is presented that these pseudoknots contain two A-form helical stems of six base-pairs each, connected by two loops, in a classic H-type pseudoknot topology. A particularly interesting feature is that the shorter of the two connecting loops, loop 1, consists of only a single adenosine nucleotide that spans the major groove of stem 2. In this respect, the frameshift-associated pseudoknots are structurally similar to the pseudoknot within the gene 32 mRNA of bacteriophage T2, previously characterized by NMR methods. Despite having similar nucleotide sequences, the solvent exchange rates of the imino protons at the junction of the helical stems in the wild-type and mutant frameshifting pseudoknots differ from each other and from the bacteriophage T2 pseudoknot. The implications of this finding are discussed.

Non-nearest neighbor effects on the thermodynamics of unfolding of a model mRNA pseudoknot.

The upstream autoregulatory mRNA leader sequence of gene 32 of 17 T-even and related bacteriophages folds into a simple tertiary structural motif, a hairpin-type RNA pseudoknot. In phage T4, the pseudoknot is contained within 28 contiguous nucleotides which adopt a pseudocontinuous helical structure derived from two coaxially stacked helical stems of four (stem 1) and seven (stem 2) base-pairs connected by two inequivalent single-stranded loops of five and one nucleotide(s). These two loops cross the minor and major grooves of stems 1 and 2, respectively. In this study, the equilibrium unfolding pathway of a 35-nucleotide RNA fragment corresponding to the wild-type and sequence variants of the T4 gene 32 mRNA has been determined through analysis of dual-wave-length, equilibrium thermal melting profiles via application of a van't Hoff model based on multiple sequential, two-state transitions. The melting profile of the wild-type RNA is well-described by two sequential melting transitions over a wide range of magnesium concentration. Compensatory base-pair substitutions incorporated into helical stems 1 and 2 were used to assign the first low enthalpy, moderate tm melting transition to the denaturation of the short three to four base-pair stem 1, followed by unfolding of the larger seven base-pair stem 2. We find that loop 1 substitution mutants (A10 to G10, C10, U10 or GA10) strikingly uncouple the melting of stems 1 and 2, with the U10 substitution and the GA10 loop expansion more destabilizing than the G10 and C10 substitutions. A significant increase in the extent of cleavage by RNase T1 following the conserved G26 (the 3' nucleotide in loop 2) in the U10, G10, and GA10 mutants suggests that an altered helix-helix junction region in this mutant may be responsible, at least in part, for this uncoupling. In addition to a modest destabilization of stem 2, the major effect of deletion or nucleotide substitution in the 3' single-stranded tail is a destabilization of stem 1, a non-nearest neighbor tertiary structural effect, which may well be transmitted through an altered loop 1-core helix interaction. In contrast, truncation of the 5' tail has no effect on the stability of the molecule.

Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic complementation groups.

This study examines the contribution of domain II beta of bacteriophage Mu transposase (A protein), a subdomain of the central catalytic domain II, to the transposition reaction. The properties of several point mutations implicate a role for this domain in facilitating metal-assisted assembly of the synaptic complex, as well as in intramolecular DNA strand transfer. Point mutations as well as deletions in domain II beta can be complemented by those in domain II alpha but not those in domain III alpha. Thus, residues within subdomains II alpha and II beta belong to different catalytic complementation groups.

Solution structure and backbone dynamics of Mason-Pfizer monkey virus (MPMV) nucleocapsid protein.

Retroviral nucleocapsid proteins (NCPs) are CCHC-type zinc finger proteins that mediate virion RNA binding activities associated with retrovirus assembly and genomic RNA encapsidation. Mason-Pfizer monkey virus (MPMV), a type D retrovirus, encodes a 96-amino acid nucleocapsid protein, which contains two Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc fingers connected by an unusually long 15-amino acid linker. Homonuclear, two-dimensional sensitivity-enhanced 15N-1H, three-dimensional 15N-1H, and triple resonance NMR spectroscopy have been used to determine the solution structure and residue-specific backbone dynamics of the structured core domain of MPMV NCP containing residues 21-80. Structure calculations and spectral density mapping of N-H bond vector mobility reveal that MPMV NCP 21-80 is best described as two independently folded, rotationally uncorrelated globular domains connected by a seven-residue flexible linker consisting of residues 42-48. The N-terminal CCHC zinc finger domain (residues 24-37) appears to adopt a fold like that described previously for HIV-1 NCP; however, residues within this domain and the immediately adjacent linker region (residues 38-41) are characterized by extensive conformational averaging on the micros-ms time scale at 25 degrees C. In contrast to other NCPs, residues 49-77, which includes the C-terminal CCHC zinc-finger (residues 53-66), comprise a well-folded globular domain with the Val49-Pro-Gly-Leu52 sequence and C-terminal tail residues 67-77 characterized by amide proton exchange properties and 15N R1, R2, and (1H-15N) NOE values indistinguishable to residues in the core C-terminal finger. Twelve refined structural models of MPMV NCP residues 49-80 (pairwise backbone RMSD of 0.77 A) reveal that the side chains of the conserved Pro50 and Trp62 are in van der Waals contact with one another. Residues 70-73 in the C-terminal tail adopt a reverse turn-like structure. Ile77 is involved in extensive van der Waals contact with the core finger domain, while the side chains of Ser68 and Asn75 appear to form hydrogen bonds that stabilize the overall fold of this domain. These residues outside of the core finger structure are conserved in D-type and related retroviral NCPs, e.g., MMTV NCP, suggesting that the structure of MPMV NCP may be representative of this subclass of retroviral NCPs.

Retroviral nucleocapsid proteins possess potent nucleic acid strand renaturation activity.

The nucleocapsid protein (NC) is the major genomic RNA binding protein that plays integral roles in the structure and replication of all animal retroviruses. In this report, select biochemical properties of recombinant Mason-Pfizer monkey virus (MPMV) and HIV-1 NCs are compared. Evidence is presented that two types of saturated Zn2 NC-polynucleotide complexes can be formed under conditions of low [NaCl] that differ in apparent site-size (n = 8 vs. n = 14). The formation of one or the other complex appears dependent on the molar ratio of NC to RNA nucleotide with the putative low site-size mode apparently predominating under conditions of protein excess. Both MPMV and HIV-1 NCs kinetically facilitate the renaturation of two complementary DNA strands, suggesting that this is a general property of retroviral NCs. NC proteins increase the second-order rate constant for renaturation of a 149-bp DNA fragment by more than four orders of magnitude over that obtained in the absence of protein at 37 degrees C. The protein-assisted rate is 100-200-fold faster than that obtained at 68 degrees C, 1 M NaCl, solution conditions considered to be optimal for strand renaturation. Provided that sufficient NC is present to coat all strands, the presence of 400-1,000-fold excess nonhomologous DNA does not greatly affect the reaction rate. The HIV-1 NC-mediated renaturation reaction functions stoichiometrically, requiring a saturated strand of DNA nucleotide:NC ratio of about 7-8, rather than 14. Under conditions of less protein, the rate acceleration is not realized. The finding of significant nucleic acid strand renaturation activity may have important implications for various events of reverse transcription particularly in initiation and cDNA strand transfer.

Metal response element (MRE)-binding transcription factor-1 (MTF-1): structure, function, and regulation.

Metal-responsive control of the expression of genes involved in metal metabolism and metal homeostasis allows an organism to tightly regulate the free or bioavailable concentration of beneficial metal ions, such as zinc, copper, and iron, within an acceptable range, while efficiently removing nonbeneficial or toxic metals. Emerging evidence also suggests that metal homeostasis is intimately coupled to the oxidative stress response in many cell types. The expression of genes that encode metallothioneins in all vertebrate cells is strongly induced by potentially toxic concentrations of zinc and cadmium, as well as in response to strong oxidizing agents, including hydrogen peroxide. This induction requires a cis-acting DNA element, termed a metal response element (MRE), and MRE-binding transcription factor-1 (MTF-1), a Cys2-His2 zinc finger protein. This review summarizes recent progress that has been made toward understanding the structure, function, and metalloregulation of mammalian MTF-1.

des-(1-13) human beta-endorphin interacts with calmodulin.

It is known that the 31-residue neuropeptide beta-endorphin inhibits the calcium-dependent, calmodulin-mediated stimulation of cyclic nucleotide phosphodiesterase activity. The results of this study demonstrate that a non-opiate, synthetic amino terminal deletion peptide, des-(1-13), of human beta-endorphin is also capable of inhibiting the stimulated enzymic activity, but not the basal activity. This inhibition occurs with the same efficacy as the intact 31-residue peptide. Thus, the amino terminal region of beta-endorphin, which is responsible for opiate activity, does not appear to contribute to the calmodulin interaction. Circular dichroic spectroscopy of des-(1-13) beta-endorphin, calmodulin, and mixtures of the two shows that the ellipticity at 221 nm was more negative in the peptide-protein mixture than could be accounted for on the basis of simple additivity of the peptide and calmodulin. This spectral change implies enhanced alpha-helicity concomitant with the peptide-protein association. Helix formation may occur in the peptide since this sequence has the potential to form an amphipathic helix.

Zinc metalloproteins involved in replication and transcription.

RNA polymerase (RPase) from E. coli contains two tightly incorporated Zn(II) ions, while the monomeric RPase from bacteriophage T7 does not contain zinc and does not require Zn(II) in the assay. One of the two Zn(II) ions can be differentially removed from E. coli RPase with p-hydroxymercuriphenylsulfonate (PMPS) combined with EDTA and thiol. The resultant Znl or ZnA RPase shows no alteration in transcription initiation and elongation rate from sigma-specific promoters. Biosynthesis of a Co2 RPase and formation of CoA RPase by similar treatment shows the tetrahedral-type Co(II) d-d absorption bands to be associated only with the Co(II) at the A site with maxima at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. Sulfur to Co(II) charge transfer bands are present at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. The absorption characteristics strongly suggest that the A site is a tetrathiolate site. While DNA polymerases do not in general appear to contain zinc, gene 32 protein (g32P) from bacteriophage T4, an accessory protein essential for DNA replication and recombination and translational control in the T4 life cycle, is a Zn(II) metalloprotein and contains 1 gram atom of tightly incorporated Zn(II). PMPS displaces the zinc by reacting with three SH groups. Apo-g32P shows markedly altered DNA binding properties. Co(II) substitution gives a protein with intense d-d transitions typical of a tetrahedral Co(II) complex with absorption maxima at 680 (epsilon = 480), 645 (epsilon = 660), 605 (epsilon = 430), 355 (epsilon = 2250), and 320 (epsilon = 3175) nm. The data support a 3 Cys, 1 His coordination site located in the middle of the DNA binding domain of g32P. Data thus far suggest that the Zn(II) binding sites in multisubunit RNA polymerases and in accessory proteins involved in polynucleotide biosynthesis are more likely to play structural or allosteric (regulatory) roles rather than directly participating in catalysis.

Structural and functional differences between the two intrinsic zinc ions of Escherichia coli RNA polymerase.

DNA-dependent RNA polymerase (RPase) from Escherichia coli contains 2 mol of intrinsic Zn(II)/mol of core enzyme (alpha 2 beta beta'). In techniques analogous to those employed with the Zn(II) metalloenzyme aspartate transcarbamoylase [Hunt, J. B., Neece, S. H., Schachman, H. K., & Ginsberg, A. (1984) J. Biol. Chem. 259, 14793-14803], we show that titration of core or holoRPase with 10 or 16 equiv, respectively, of the sulfhydryl reagent p-(hydroxymercuri)benzenesulfonate (PMPS) results in the facile release of 1 mol of Zn(II) [B-site Zn(II)] in a reaction totally reversible with the addition of excess thiol provided no metal chelator is present. If ethylenediaminetetraacetic acid (EDTA) is present, reversal of the PMPS-enzyme complex results in formation of a Zn1 RPase [A-site Zn(II)]. This enzyme retains full transcriptional activity relative to Zn2 RPase on both calf thymus (nonspecific) and T7 (sigma-dependent, specific) DNA templates. If the core enzyme-PMPS complex is incubated with a large excess of another metal such as Cd(II) followed by thiol treatment, a hybrid ZnACdB RPase is formed. Direct treatment of the enzyme with excess Cd(II) also gives rise to a hybrid ZnACdB RPase. Transcription by these enzymes is also comparable to that of the starting Zn2 enzyme. Isolation of in vivo synthesized Co2 RPase and Cd2 RPase and treatment of either enzyme with PMPS/EDTA results in formation of a CoA and CdA enzyme, respectively. Co(II)A and Cd(II)A enzymes show 123 and 76%, respectively, of the elongation rates on T7 DNA observed for the Zn(II) enzyme. Visible absorption spectroscopy of the Co2 enzyme exhibits four d-d transition bands positioned at 760 (epsilon = 800), 710 (epsilon = 900), 602 (epsilon = 1500), and 484 (epsilon = 4000) nm. In addition, two charge-transfer bands are found at 350 (epsilon = 9600) and 370 (epsilon = 9500) nm. Only the Co(II) ion bound at site A is associated with this unique set of intense d-d transitions. The positions and intensities of both the visible and charge-transfer bands of Co(II)A RPase approximate those shown by Co(II)-substituted metalloenzyme sites where the ligands are four S rather than mixed S,N or S,O sites.

Energetics of arginine-4 substitution mutants in the N-terminal cooperativity domain of T4 gene 32 protein.

Gene 32 protein (gp32) from bacteriophage T4 is a sequence-nonspecific single-strand (ss) nucleic acid binding protein which binds highly cooperatively to ss nucleic acids. The N-terminal "B" or basic domain (residues 1-21) is known to be required for highly cooperative binding by gp32 (where K(app) = K(int) omega, omega > or = 500), since its removal results in a protein which binds ss nucleic acids noncooperatively (omega = 1). In this paper, we probe the molecular details of cooperative binding by gp32 by physicochemical characterization of a set of four single amino acid substitution mutants of Arg4: Lys4 (R4K gp32), Gln4 (R4Q gp32), Thr4 (R4T gp32), and Gly4 (R4G gp32). The qualitative ranking of binding affinities to poly(A) is wild-type > or = R4K > R4Q > R4T > R4G > gp32-B (gp32 lacking the first 21 amino acids). The occluded site size is n(app) = 7.5 +/- 0.5 for all gp32s. Resolution of K(int) and omega for wild-type, R4K, R4Q, and R4T gp32s was estimated under conditions of low lattice saturation (v < or = 0.011) using multiple reverse fluorescence titrations collected at 10 mM Tris-HCl, pH 8.1, 20 degrees C, and a NaCl concentration where K(app) was (2-4) x 10(6) M-1 for each gp32 on the ribohomopolymer poly(A). Binding parameters for all gp32s were obtained directly or compared by conservative extrapolation of the [NaCl] dependence of K(app) to 0.20 M NaCl, 20 degrees C, pH 8.1. The magnitude of omega was then assumed not to vary with [NaCl] (shown for R4T gp32), allowing estimation of K(int) at 0.20 M NaCl. We find that R4K gp32 binds to poly(A) with an overall affinity (K(app)) which is 2-3-fold lower than wild-type gp32, while omega for each molecule seems indistinguishable (wild-type gp32, omega approximately 800-1300; R4K gp32, omega approximately 600-1200). Surprisingly, R4Q gp32 is characterized by an omega also not readily distinguishable from the wild-type and R4K proteins (omega approximately 800-4400), while K(app) is reduced about 10-fold. This mutant also shows a significantly reduced [NaCl] dependence of the binding to poly(A). R4T gp32 binds about 10-fold weaker than the Q mutant. It exhibits an omega ranging from 300 to 700 and a substantially reduced [NaCl] dependence (delta log K(int)/delta log [NaCl] = -1.4 from 0.10 to 0.20 M NaCl), indicative of significant perturbations in both K(int) and omega terms.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of substitution of proposed Zn(II) ligand His81 or His64 in phage T4 gene 32 protein: spectroscopic evidence for a novel zinc coordination complex.

T4 gene 32 protein (gp32), the prototype helix-destabilizing or single-stranded (ss) DNA binding protein, contains one tightly coordinated Zn2+ ion bound tetrahedrally by three cysteines (residues 77, 87, and 90) and a fourth non-thiol donor. In previous work, it was shown that the proposed non-thiol ligand His81 could be readily substituted with nonliganding glutamine and alanine residues without deleterious effects on gp32 structure and simple assays of ssDNA binding. In this paper we show that exchange broadening of bulk 35Cl- anion by protein-bound Zn(II) is not observed in the His81-->Ala (H81A) mutant, unless the coordination site is disrupted with an organomercurial, p-mercuriphenylsulfonate. This suggests that, in the mutant protein, anions, and by implication solvent molecules, do not gain access to a newly formed inner shell Zn(II) coordination site as a result of mutagenesis. H81A gp32 is characterized by nearly wild-type helix-destabilizing activity on poly(d[A-T]) and highly cooperative binding to the polynucleotide poly(A) at pH 7.7 over the temperature range from 20 to 42 degrees C at 0.35 M NaCl, exhibiting only a approximately 2.5-4-fold decrease in poly(A) affinity. Limited proteolysis experiments show that an additional tryptic cleavage site maps to the Arg111-Lys112 bond within the protease-resistant core domain of the H81A gp32 following long incubation times and results in the accumulation of a 16-kDa subcore fragment. This new cleavage site is within the internal LAST motif, which has been proposed to be directly involved in cooperative ssDNA binding [Casas-Finet, J. R., & Karpel, R. L. (1993) Biochemistry 32, 9735-9744]. Thus substitution of His81 with Ala subtly alters the conformation or dynamics of the backbone around the LAST motif, which may be manifest as a moderately lower cooperative binding affinity of H81A gp32 for polynucleotides. H81A gp32, however, is fully functional in stimulating in vitro homologous pairing catalyzed by the T4 recombinase uvsX protein. Since substitution of His81 with a nonliganding Ala is nearly silent, we propose an alternative mode of Zn(II) coordination in T4 gene 32 protein, involving His64 rather than His81 as the fourth non-thiol ligand. That replacement of His64, and not His81, with Cys results in marked changes in the first coordination sphere of ligands as evidenced by the optical spectrum of Co(II)-substituted H64C gp32 is consistent with the noninvolvement of His81 and implicates a novel His64-X12-Cys77-X9-Cys87-X2-Cys90 coordination motif, unique among zinc-containing nucleic acid binding proteins.

Nucleic acid binding properties of recombinant Zn2 HIV-1 nucleocapsid protein are modulated by COOH-terminal processing.

The nucleocapsid protein (NC) of all animal retroviruses is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. In a previous report, we showed that recombinant NC protein from HIV-1, a 71-amino-acid protein (NC71), is apparently able to form two types of protein-nucleic acid complexes under low [NaCl], pH 8.3 and 25 degrees C. These appeared to differ in occluded apparent site size, napp, forming n = 8 and n = 14 complexes on poly(A) (Dib-Hajj, F., Khan, R., and Giedroc, D. P. (1993) Protein Sci. 2, 331-243) under conditions of high and low protein-nucleotide ratios, respectively. Here we show that both NC71-poly(A) complexes strongly scatter light under these solution conditions. Examination of the wavelength dependence of the light scattering at lambda < or = 320 nm indicates that each complex is characterized by a different scattering coefficient. Optical density measurements suggest that upon formation of the saturated n = 8 complex, additional polynucleotide is not incorporated into the complex over a period of hours, i.e. the n = 14 complex is not formed via redistribution of the n = 8 complex under low salt conditions, 25 degrees C. In contrast, the n = 14 complex readily incorporates additional protein until that sufficient to form the n = 8 complex is present. The n = 14 complex efficiently precipitates poly(A) and shows spectral characteristics expected for an extensively charge-neutralized nucleic acid complex. At [NC71] in excess of that required to form the n = 8 complex, this n = 14 complex is best described as a kinetic intermediate on the pathway to the n = 8 complex, which forms over a period of hours under low salt conditions, 25 degrees C. This slow kinetics of binding provides a possible explanation for the finding that the previously observed moderate cooperativity of Zn2 NC71 binding to poly(A) (omega = 200) at pH 8.3 and 0.29 M NaCl (Khan, R., and Giedroc, D. P. (1992) J. Biol. Chem. 267, 6689-6695) is shown here to represent a nonequilibrium phenomenon, apparently converting to a low or no cooperativity complex over a period of hours. Proteolytic removal of the COOH-terminal 14 amino acids from NC71, forming a 57-amino-acid protein (denoted NC57), removes this apparent binding site size heterogeneity of NC71 on poly(A). At 20 mM NaCl, NC57 binds with n = 6-7 nucleotides, in a manner which is independent of the protein-poly(A) nucleotide ratio. The implications of these findings on processing of the gag precursor which leads to mature NC in HIV-1 virions is discussed.

Recombinant human immunodeficiency virus type 1 nucleocapsid (NCp7) protein unwinds tRNA.

The nucleocapsid protein (NC) of all animal retroviruses, encoded by the gag gene, is the major structural protein of the core ribonucleoprotein complex, bound to genomic RNA in mature virions. NC is also thought to play one or more accessory roles in reverse transcription. Mature NC (p7NC) from human immunodeficiency virus type 1 (HIV-1) is a 71-amino acid, basic protein which contains two Cys3His Zn(II) retroviral-type zinc finger domains. Herein, we describe the subcloning and expression of HIV-1 NC, denoted NC71, from an inducible phage T7 RNA polymerase promoter in Escherichia coli. Purified NC71 can be reversibly reconstituted with 2 g.at Zn(II) determined by atomic absorption. Ultraviolet circulation dichroism spectroscopy has been used to characterize the complexes between highly purified NC71 and the RNA homopolynucleotide poly(A) and E. coli tRNA(mixed). On poly(A), Zn2 NC71 is characterized by an apparent site size n = 15 +/- 3 nucleotides and high affinity (Kapp = 3 x 10(7) M-1) and moderately cooperative (omega approximately 170 +/- 25) binding. A mixture of E. coli tRNA species (tRNA(mixed) was used to probe the conformational changes induced in tRNA upon binding of HIV-1 NC71. Two structural forms of tRNA(mixed), which differ in their degree of tertiary structure, were assayed for their susceptibility to denaturation by NC71. Five molar monomer equivalents of NC71 are required to denature the "inactive" tRNA in the absence of Mg2+. A Zn(II)-free, oxidized form of NC71 was also shown to unwind inactive tRNA with the same efficiency and stoichiometry. The detailed spectral changes which occur on NC-induced denaturation closely mimic temperature-induced denaturation of inactive tRNA(mixed). The prototype helix-destabilizing protein, T4 gene 32 protein, is unable to unwind this form of tRNA under the same conditions. The stoichiometry of unwinding of inactive tRNA by NC71 is consistent with the site size determined with poly(A). An "active" form of tRNA(mixed), prepared by thermal denaturation and refolding of the inactive form with Mg2+, proved less susceptible to both temperature and NC71-induced unwinding. The mechanistic implications of these findings on the reported biochemical activities of RNA:RNA annealing and replication primer tRNA positioning by NC are discussed.

Zinc site redesign in T4 gene 32 protein: structure and stability of cobalt(II) complexes formed by wild-type and metal ligand substitution mutants.

Phage T4 gene 32 protein (gp32) is a zinc metalloprotein which binds cooperatively and preferentially to single-stranded nucleic acids and functions as a replication and recombination accessory protein. Zn(II) coordination by gp32 employs a His-Cys3 metal ligand donor set derived from the His64-X12-Cys77-X9-Cys87-X2-Cys90 sequence in the ssDNA-binding core domain of the molecule. Crystallographic studies reveal that His64 and Cys77 are derived from two independent beta-strands within a distorted three-stranded beta-sheet and are relatively more buried from solvent than are Cys87 and Cys90, which are positioned immediately before and within, respectively, an alpha-helix. In an effort to understand the origin of the stability of the metal complex, we have employed an anaerobic optical spectroscopic, competitive metal binding assay to determine the coordination geometry and association constants (Ka) for the binding of Co(II) to wild-type gp32 and a series of zinc ligand substitution mutants. At pH 7.5, 25 degrees C, wild-type gp32 binds Co(II) with a Ka approximately 1 x 10(9) M-1. Competition experiments reveal that Ka for Zn(II) is 3.0 (+/-1.0) x 10(11) M-1. We find that all non-native metal complexes retain tetrahedral or distorted tetrahedral coordination geometry but are greatly destabilized in a manner essentially of whether a new protein-derived coordination bond is formed (e.g., in H64C gp32) or not. Co(II) binding isotherms obtained for three His64 substitution mutants, H64C, H64D, and H64N gp32s, suggest that each mutant forms a dimeric Cys4 tetrathiolate intermediate complex at limiting [Co(II)]f, each then rearranges at high [Co(II)]f to form a monomolecular site of the expected geometry and Ka approximately 1 x 10(4) M-1. Like the His64 mutants, C77A gp32 appears to form at least two types of complexes over the course of a Co(II) titration: one with octahedral coordination geometry formed at low [Co(II)]f, with a second tetrahedral or five-coordinate site formed at higher [Co(II)]f. Apo C87S and C90A gp32s, in contrast, each form a single complex at all [Co(II)]f, consistent with Cys2-His-H2O tetrahedral geometry of Ka approximately (1-2) x 10(5) M-1. These studies reveal that the local protein structure restricts accommodation of a non-native metal complex in a ligand-specific manner. The implications of this work for de novo design of zinc complexes in proteins are discussed.

Characterization of a cooperativity domain mutant Lys3 --> Ala (K3A) T4 gene 32 protein.

The N-terminal "B" domain of T4 gene 32 protein contains a Lys3-Arg4-Lys5 sequence that has been postulated to provide a major determinant for cooperative binding. In this report, the equilibrium binding properties of a Lys3 --> Ala substitution mutant of gp32 (K3A gp32) and described and compared to a set of substitution mutants of Arg4 previously described (Villemain, J. L., and Giedroc, D. P. (1993) Biochemistry 32, 11235-11246) and further characterized here. K3A gp32 exhibits binding behavior which mirrors that of R4Q gp32. Despite an 6-8-fold decrease in overall binding affinity (Kapp = Kint x omega) at pH 8.1, 0.20 M NaCl, 20 degrees C, the magnitude of the cooperativity parameter is at most 2-3-fold smaller than that of the wild-type protein. The magnitude of omega is independent of salt concentration and type over the range in [NaCl] from 0.125 to 0. 225 M and [NaF] between 0.20 and 0.32 M (log omega = 2.86 +/- 0.19). For comparison, log omega for wild-type gp32 is 2.91 (+/- 0.21) resolved at 0.275 M NaCl and 3.39 +/- 0.11 in [NaF] between 0.40 and 0.45 M. In contrast to omega, the [NaCl] dependence of Kapp is large and markedly nonlinear for both wild-type and K3A gp32s over a [NaCl] range extending from 0.05 M to 0.40 M NaCl. Modeling of the complete salt dependence of Kapp for wild-type, K3A, and R4T gp32s in NaCl and NaF with a simple ion-exchange model suggests that substitutions within the basic Lys3-Arg4-Lys5 sequence do not strongly modulate the net displacement of cations and anions upon poly(A) complex formation by gp32.

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus.

The mouse mammary tumor virus (MMTV) gag-pro frameshifting pseudoknot is an H-type RNA pseudoknot that contains an unpaired adenosine (A14) at the junction of the two helical stems required for efficient frameshifting activity. The thermodynamics of folding of the MMTV vpk pseudoknot have been compared with a structurally homologous mutant RNA containing a G x U to G-C substitution at the helical junction (U13C RNA), and an A14 deletion mutation in that context (U13CdeltaA14 RNA). Dual wavelength optical melting and differential scanning calorimetry reveal that the unpaired adenosine contributes 0.7 (+/-0.2) kcal mol(-1) at low salt and 1.4 (+/-0.2) kcal mol(-1) to the stability (deltaG(0)37) at 1 M NaCl. This stability increment derives from a favorable enthalpy contribution to the stability deltadeltaH = 6.6 (+/-2.1) kcal mol(-1) with deltadeltaG(0)37 comparable to that predicted for the stacking of a dangling 3' unpaired adenosine on a G-C or G x U base pair. Group 1A monovalent ions, NH4+, Mg2+, and Co(NH3)6(3+) ions stabilize the A14 and deltaA14 pseudoknots to largely identical extents, revealing that the observed differences in stability in these molecules do not derive from a differential or specific accumulation of ions in the A14 versus deltaA14 pseudoknots. Knowledge of this free energy contribution may facilitate the prediction of RNA pseudoknot formation from primary nucleotide sequence (Gultyaev et al., 1999, RNA 5:609-617).