On the interaction of mitochondrial complex III with the Rieske iron-sulfur protein (subunit V).

Limited proteolysis of solubilized beef heart mitochondrial complex III with trypsin yields a product previously identified as fragment V" (González-Halphen, D., Lindorfer, M. A., and Capaldi, R. A. (1988) Biochemistry 27, 7021-7031). In this work, fragment V" was generated by trypsin treatment of both the intact complex III and the purified Rieske iron-sulfur protein. Thus, in its bound or isolated form, the same sites of subunit V are sensitive to protease action. Fragment V" was a soluble protein that retained its iron-sulfur moiety. It was purified by exclusion from a hydrophobic phenyl-Sepharose CL-4B column followed by gel filtration. In contrast to the pure, intact subunit V, fragment V" did not reconstitute oxidoreductase activity when combined with complex III devoid of subunit V. However, a 20-amino acid synthetic peptide carrying the sequence between amino acids Lys33 and Lys52 of the Rieske iron-sulfur protein competed with intact subunit V in reconstitution assays. The results obtained suggest that the iron-sulfur protein binds to complex III by hydrophobic protein-protein interactions, and that a nontransmembrane 18-amino acid amphipathic stretch accounts for the association of this subunit to the rest of the complex.     

Identification of paramyxovirus V protein residues essential for STAT protein degradation and promotion of virus replication

Some paramyxovirus V proteins induce STAT protein degradation, and the amino acids essential for this process in the human parainfluenza virus type 2 (hPIV2) V protein have been studied. Various recombinant hPIV2s and cell lines constitutively expressing various mutant V proteins were generated. We found that V proteins with replacement of Cys residues of the Cys cluster were still able to bind STATs but were unable to induce their degradation. The hPIV2 V protein binds STATs via a W-(X)3-W-(X)9-W Trp motif located just upstream of the Cys cluster. Replacements of two or more Trp residues in this motif resulted in a failure to form a V/STAT2 complex. We have also identified two Phe residues of the hPIV2 V protein that are essential for STAT degradation, namely, Phe207, lying within the Cys cluster, and Phe143, in the P/V common region of the protein. Interestingly, infection of BHK cells with hPIV2 led to the specific degradation of STAT1 and not STAT2. Other evidence for the cell species specificity of hPIV2-induced STAT degradation is presented. Finally, a V-minus hPIV2, which can express only the P protein from its P gene, was generated and partially characterized. In contrast to V-minus viruses of other paramyxovirus genera, this V-minus rubulavirus was highly debilitated, and its growth even in Vero cells was very limited. The structural rubulavirus V proteins, as expected, are thus clearly important in promoting virus growth, independent of their anti-interferon (IFN) activity. Interestingly, many of the residues that are essential for anti-IFN activity, e.g., the Cys of this cluster and Phe207 within this cluster, as well as the Trp of this motif, are also essential for promoting virus growth.     

Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination

Mutations in the Artemis protein in humans result in hypersensitivity to DNA double-strand break-inducing agents and absence of B and T lymphocytes (radiosensitive severe combined immune deficiency [RS-SCID]). Here, we report that Artemis forms a complex with the 469 kDa DNA-dependent protein kinase (DNA-PKcs) in the absence of DNA. The purified Artemis protein alone possesses single-strand-specific 5' to 3' exonuclease activity. Upon complex formation, DNA-PKcs phosphorylates Artemis, and Artemis acquires endonucleolytic activity on 5' and 3' overhangs, as well as hairpins. Finally, the Artemis:DNA-PKcs complex can open hairpins generated by the RAG complex. Thus, DNA-PKcs regulates Artemis by both phosphorylation and complex formation to permit enzymatic activities that are critical for the hairpin-opening step of V(D)J recombination and for the 5' and 3' overhang processing in nonhomologous DNA end joining.     

Computational anti-AIDS drug design based on the analysis of the specific interactions between immunophilins and the HIV-1 gp120 V3 loop. Application to the FK506-binding protein

The object of the study was to model the structural complex of the FK506-binding protein (FKBP) with the CRK peptide imitating the central region of the HIV-1 V3 loop, as well as to define the FKBP stretch giving rise to the binding site for V3 the synthetic copy of which, on the assumption of preserving the spatial peptide structure in the free state, can be considered as a promising applicant for the role of antiviral drug. To this end, the following successive steps were carried out: (i) the NMR-based conformational analysis of CRK was put into practice, and, in the light of the results derived, the best energy CRK structure meeting the requirements of the input NMR data was identified; (ii) molecular docking of the CRK structure with the X-ray FKBP conformation was implemented, and energy refining the simulated structural complex was realized; (iii) the matrix of distances between amino acids of the ligand and receptor was computed to specify the FKBP stretch keeping in touch with CRK followed by analyzing the types of interactions stabilizing the over-molecular ensemble; (iv) 3D structure of this stretch in the unbound status referred to as the FKBP peptide was predicted, and its collation with the X-ray conformation of the identical FKBP site was performed; (v) the potential energy function and its constituents were studied for the structural complex generated by molecular docking of the CRK molecule with the FKBP peptide; and (vi) from all evidence, the virtual FKBP-derived peptide was submitted to be utilized as a prospective structural framework in the anti-HIV-1 drug design. Summing up the results obtained, the following principal conclusion was drawn: a high affinity of the V3 loop peptide to the FKBP is based on the principle of "mirror similarity" that implies the near resemblance of 3D structures for the two individual fragments of the receptor and ligand, which, most likely, accounts for recognizing the immunophilin by V3 and determines the specificity of their efficacious interactions arising from the experimental observations.     

Immunophilins and HIV-1 V3 Loop For Structure-Based Anti-AIDS Drug Design

Abstract

The model of the structural complex of cyclophilin A (CycA) belonging to the immunophilins family with the HIV-MN gpl20 V3 loop was generated, and the computer-aided design of the immunophilin-derived peptide able to mask the biologically crucial V3 segments was implemented.

To this end, the following problems were solved: (i) the NMR-based conformational analysis of the HIV-MN V3 loop was put into effect, and its low energy structure fitting the input experimental observations was determined; (ii) molecular docking of this V3 structure with the X-ray conformation of CycA was carried out, and the energy refining the simulated structural complex was performed; (iii) the matrix of inter-atomic distances for the amino acids of the molecules forming part of the built over-molecular ensemble was computed, the types of interactions responsible for its stabilization were analyzed, and the CycA stretch, which accounts for the binding to V3, was identified; (iv) the most probable 3D structure for this stretch in the unbound state was predicted, and its collation with the X-ray structure for the corresponding site of CycA was performed; (v) the potential energy function and its constituents were studied for the structural complex generated by molecular docking of the V3 loop with the CycA peptide offering the virtual molecule that imitates the CycA segment, making a key contribution to the interactions of the native protein with the HIV-1 principal neutralizing determinant; (vi) as a result of the studies above, the designed molecule was shown to be capable of the efficacious blockading the functionally crucial V3 sites; and (vii) based on the joint analysis of the evidence obtained previously and in the present study, the composition of the peptide cocktail presenting the promising anti-AIDS pharmacological substance was developed.

The molecules simulated here by molecular modeling methods may become the first representatives of a new class of the chemical compounds (immunophilin-derived peptides) offering the looking-forward basic structures for the design of efficacious and safe antiviral agents.     

Identification of the 37-kDa protein displaying a variable interaction with the erythroid cell membrane as glyceraldehyde-3-phosphate dehydrogenase

In previous studies from this laboratory we isolated and characterized a 37-kDa protein that was associated with the membrane of erythroid cells. The polypeptide appeared to undergo a lineage-specific alteration in its interaction with the membrane during erythroid development and migrated as a family of isoelectric focusing variants during analyses on two-dimensional gels. We report here that the 37-kDa protein is homologous to the enzyme glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). This conclusion was reached from the results of several experimental approaches comparing the biochemical and genetic properties of the 37-kDa protein (p37) with authentic glyceraldehyde-3-phosphate dehydrogenase. Peptide maps of highly purified p37 and glyceraldehyde-3-phosphate dehydrogenase, generated with Staphylococcus V8 protease, were identical. The nucleotide sequence of a cDNA clone encoding p37 was nearly identical to the published sequence for genes encoding glyceraldehyde-3-phosphate dehydrogenase. These results suggest that the interaction of the enzyme with the red cell membrane is more complex than previously envisioned. The existence of subpopulations of glyceraldehyde-3-phosphate dehydrogenase molecules is envisioned that exhibit different levels of enzyme activity and bind to the red cell membrane with varying affinities.     

A study on the mechanism of the vanadate-dependent NADH oxidation

The mechanism of the vanadate (V(_v))-dependent oxidation of NADH was different in phosphate buffers and in phosphate-free media. In phosphate-free media (aqueous medium or HEPES buffer) the vanadyl (V(_v)) generated by the direct V(_v)-dependent oxidation of NADH formed a complex with V(_v). In phosphate buffers V(_v) autoxidized instead of forming a complex with V(_v). The generated superoxide radical (O₂⁻) initiated, in turn, a high-rate free radical chain oxidation of NADH. Phosphate did not stimulate the V(_v)-dependent NADH oxidation catalyzed by O₂⁻-generating systems. Monovanadate proved to be a stronger catalyzer of NADH oxidation as compared to polyvanadate.     

A mutational analysis reveals new functional interactions between domains of the Oxa1 protein in Saccharomyces cerevisiae

The Oxa1/YidC/Alb3 family plays a key role in the biogenesis of the respiratory and photosynthetic complexes in bacteria and organelles. In Saccharomyces cerevisiae, Oxa1 mediates the co‐translational insertion of mitochondrially encoded subunits of the three respiratory complexes III, IV and V within the inner membrane and also controls a late step in complex V assembly. No crystal structure of YidC or Oxa1 is available and little is known about the respective role of each transmembrane segment (TM) and hydrophilic loop of this polytopic protein on the biogenesis of the three complexes. Here, we have generated a collection of random point mutations located in the hydrophobic and hydrophilic domains of the protein and characterized their effects on the assembly of the three respiratory complexes. Our results show mutant‐dependent differential effects, particularly on complex V. In order to identify tertiary interactions within Oxa1, we have also isolated revertants carrying second‐site compensatory mutations able to restore respiration. This analysis reveals the existence of functional interactions between TM2 and TM5, TM4 and TM5 as well as between TM4 and loop 2, highlighting the key position of TM4 and TM5 in the Oxa1 protein.     

In vitro processing of the 3'-overhanging DNA in the postcleavage complex involved in V(D)J joining

The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3'-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3'-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3'-processing activity is observed not only with the core RAG2 but also with the full-length protein.     

Characterization of Polyoma DNA-Protein Complexes I. Electrophoretic Identification of the Proteins in a Nucleoprotein Complex Isolated from Polyoma-Infected Cells

A study was undertaken to examine polyoma DNA-protein complexes. A biophysical characterization of the complexes was made, and the proteins found in such complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison was made between a 52S nucleoprotein complex isolated from nuclei of 26-h polyoma-infected cells and a 28S virion core complex ejected out of mature virus particles. It was found that both complexes were reduced to a 20S viral DNA component plus free protein after incubation in 1 M NaCl or Sarkosyl. Treatment of the complexes with either Pronase or 0.5 M NaCl resulted in only partial removal of proteins from the viral DNA. After fixation in formaldehyde, the 52S nucleoprotein complex had a buoyant density of 1.45 g/cm³, and the virion core complex had a buoyant density of 1.59 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel profiles of purified polyoma virion proteins, used as a reference marker, demonstrated three capsid proteins, V1 to V3, as well as four histones, V4 to V7, which constituted about 7% of the total virion protein. Electrophoretic analysis of the proteins comprising the 52S nucleoprotein complex revealed that the same seven proteins present in the mature virion were also found in this complex. However, the ratios of the proteins in the complex were quite different from that of the mature virion, with the four histones comprising 48% of the total complex protein. A pulse-chase experiment of the nucleoprotein complex demonstrated that the 26-h complex was chased into mature virions.     

Co-immunization with multimeric scaffolds and DNA rapidly induces potent autologous HIV-1 neutralizing antibodies and CD8+ T cells

To obtain proof of concept for HIV vaccines, we generated recombinant multimeric particles displaying the HIV-1 Envelope (Env) third hypervariable region (V3) as an N-terminal fusion protein on the E2 subunit of the pyruvate dehydrogenase complex of Geobacillus stearothermophilus. The E2 scaffold self-assembles into a 60-mer core that is 24 nm in diameter, with a molecular weight of 1.5 MDa, similar to a virus like particle with up to 60 copies of a heterologous protein accessible on the surface. Env(V3)-E2 multimers were tested alone and in combination with Env(gp160) DNA in mice and rabbits. Following two or more co-immunizations with Env(V3)-E2 and Env gp160 DNA, all 18 rabbits developed potent autologous neutralizing antibodies specific for V3 in six weeks. These neutralizing antibodies were sustained for 16 weeks without boosting, and comparable responses were obtained when lipopolysaccharide, a contaminant from expression in E. coli, was removed. Co-immunizations of Env(V3)-E2 and DNA expressing gp160 elicited moderate CD8-specific responses and Env-specific antibodies in mice. Co-immunization with DNA and E2 was superior to individual or sequential vaccination with these components in eliciting both neutralizing antibodies in rabbits and CD8(+) T cell responses in mice. Co-immunization with DNA and multimeric E2 scaffolds appears to offer a highly effective means of eliciting rapid, specific, and sustained immune responses that may be a useful approach for other vaccine targets.     

The C‐terminal portion of the cleaved HT motif is necessary and sufficient to mediate export of proteins from the malaria parasite into its host cell

The malaria parasite exports proteins across its plasma membrane and a surrounding parasitophorous vacuole membrane, into its host erythrocyte. Most exported proteins contain a Host Targeting motif (HT motif) that targets them for export. In the parasite secretory pathway, the HT motif is cleaved by the protease plasmepsin V, but the role of the newly generated N‐terminal sequence in protein export is unclear. Using a model protein that is cleaved by an exogenous viral protease, we show that the new N‐terminal sequence, normally generated by plasmepsin V cleavage, is sufficient to target a protein for export, and that cleavage by plasmepsin V is not coupled directly to the transfer of a protein to the next component in the export pathway. Mutation of the fourth and fifth positions of the HT motif, as well as amino acids further downstream, block or affect the efficiency of protein export indicating that this region is necessary for efficient export. We also show that the fifth position of the HT motif is important for plasmepsin V cleavage. Our results indicate that plasmepsin V cleavage is required to generate a new N‐terminal sequence that is necessary and sufficient to mediate protein export by the malaria parasite.     

Sequence variability in the retinal-attachment domain of mammalian rhodopsins.

Ovine rhodopsin was regenerated with 11-cis-[15-3H]retinal and cleaved in situ by Staphylococcus aureus V8 proteinase to give two membrane-bound fragments of Mr 27 000 (V8-L) and 12 000 (V8-S). After purification of the proteolysed complex by affinity chromatography with concanavalin A-Sepharose 4B, [3H]retinal was covalently linked to the protein by reduction with borohydride. The purified [3H]-retinyl V8-S fragment was cleaved with CNBr and trifluoroacetic acid, the resulting peptides resolved by gel filtration and the [3H]retinyl peptide sequenced. The protocol developed for the isolation and sequencing of this region of the ovine protein was applied directly, and reproducibly, to bleached and unregenerated porcine and equine opsins. Comparisons of the primary structures of the fragments reveals marked variation in the sequence immediately after the lysine residue shown in the ovine protein to be the attachment point for the aldehyde group of the chromophore. Mutable positions are localized in regions previously predicted as adopting nonregular or distorted conformations and hint at structural arrangements that may provide a better understanding of the spectral and functional properties of the visual pigment.     

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.     

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.     

Macrophage-tropic human immunodeficiency virus isolates from different patients exhibit unusual V3 envelope sequence homogeneity in comparison with T-cell-tropic isolates: definition of critical amino acids involved in cell tropism.

Previous experiments indicate that the V3 hypervariable region of the human immunodeficiency virus (HIV) envelope protein influences cell tropism of infection; however, so far no consistent V3 sequence can account for macrophage or T-cell tropism. In these experiments, we studied infectious recombinant HIV clones constructed by using V3 region sequences of HIV isolates from 16 patients to search for sequences associated with cell tropism. Remarkable homology was seen among V3 sequences from macrophage-tropic clones from different patients, and a consensus V3 region sequence for patient-derived macrophage-tropic viruses was identified. In contrast, V3 sequences of T-cell-tropic clones from different patients were highly heterogeneous, and the results suggested that sequence diversity leading to T-cell tropism might be generated independently in each patient. Site-specific mutations identified amino acids at several positions on each side of the GPGR motif at the tip of the V3 loop as important determinants of tropism for T cells and macrophages. However, a wide variety of mutant V3 sequences induced macrophage tropism, as detected in vitro. Therefore, the homogeneity of macrophage-tropic patient isolates appeared to be the result of selection based on a biological advantage in vivo.     

Identification of the catalytic nucleophile of tRNA (m5U54)methyltransferase

A covalent complex between tRNA (m5U54)methyltransferase, 5-fluorouridine tRNA(Phe), and S-adenosyl-L-[methyl-3H]methionine was formed in vitro and purified. Previously, it was shown that in this complex the 6-position of fluorouridine-54 is covalently linked to a catalytic nucleophile and the 5-position is bound to the transferred methyl group of AdoMet [Santi, D. V., & Hardy, L. W. (1987) Biochemistry 26, 8599-8606]. Proteolysis of the complex generated a [3H]methyl-FUtRNA-bound peptide, which was purified by 7 M urea-15% polyacrylamide gel electrophoresis. The peptide component of the complex was sequenced by gas-phase Edman degradation and found to contain two cysteines. The tritium was shown to be associated with Cys 324 of the methyltransferase, which unequivocally identifies this residue as the catalytic nucleophile.     

Simian virus 5 V protein acts as an adaptor, linking DDB1 to STAT2, to facilitate the ubiquitination of STAT1

The V protein of simian virus 5 (SV5) facilitates the ubiquitination and subsequent proteasome-mediated degradation of STAT1. Here we show, by visualizing direct protein-protein interactions and by using the yeast two-hybrid system, that while the SV5 V protein fails to bind to STAT1 directly, it binds directly and independently to both DDB1 and STAT2, two cellular proteins known to be essential for SV5-mediated degradation of STAT1. We also demonstrate that STAT1 and STAT2 interact independently of SV5 V and show that SV5 V protein acts as an adaptor molecule linking DDB1 to STAT2/STAT1 heterodimers, which in the presence of additional accessory cellular proteins, including Cullin 4a, can ubiquitinate STAT1. Additionally, we show that the avidity of STAT2 for V is relatively weak but is significantly enhanced by the presence of both STAT1 and DDB1, i.e., the complex of STAT1, STAT2, DDB1, and SV5 V is more stable than a complex of STAT2 and V. From these studies we propose a dynamic model in which SV5 V acts as a bridge, bringing together a DDB1/Cullin 4a-containing ubiquitin ligase complex and STAT1/STAT2 heterodimers, which leads to the degradation of STAT1. The loss of STAT1 results in a decrease in affinity of binding of STAT2 for V such that STAT2 either dissociates from V or is displaced from V by STAT1/STAT2 complexes, thereby ensuring the cycling of the DDB1 and SV5 V containing E3 complex for continued rounds of STAT1 ubiquitination and degradation.     

Stimulus-induced phosphorylation of vacuolar H(+)-ATPase by protein kinase A

Eukaryotic vacuolar-type H(+)-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V(1)V(0) holoenzyme into a cytosolic V(1) complex and a membrane-bound V(0) complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V(1) subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V(1) complex but not as a part of the V(1)V(0) holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V(1) complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V(1)V(0) holoenzyme.