Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.     

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.     

Dimerization and ion binding properties of S100P protein

Functional S100P requires dimer formation and dimerization might form for one of the two reasons: i. producing a pair of sites for target protein binding or ii. modulation of cation binding affinity. The extent of exposed protein hydrophobicity was related to dimer formation.     

Mapping by in vitro constructs of the P100gag-mil region, accounting for induction of chicken neuroretina cell proliferation.

The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.     

Molecular cloning of the avian acute transforming retrovirus MH2 reveals a novel cell-derived sequence (v-mil) in addition to the myc oncogene

Mill-Hill-2 virus (MH2) proviral DNA was cloned from a transformed non-producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2-specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v-mil, from MilI-Hill-2 virus, is not homologous to v-myc, the previously described oncogene of MH2, nor to avian leukaemia virus-related sequences. Evidence is presented here that v-mil has a cellular counterpart (c-mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.     

Conformational and thermodynamic properties of peptide binding to the human S100P protein

S100P is a member of the S100 subfamily of calcium-binding proteins that are believed to be associated with various diseases, and in particular deregulation of S100P expression has been documented for prostate and breast cancer. Previously, we characterized the effects of metal binding on the conformational properties of S100P and proposed that S100P could function as a Ca2+ conformational switch. In this study we used fluorescence and CD spectroscopies and isothermal titration calorimetry to characterize the target-recognition properties of S100P using a model peptide, melittin. Based on these experimental data we show that S100P and melittin can interact in a Ca2+-dependent and -independent manner. Ca2+-independent binding occurs with low affinity (Kd approximately 0.2 mM), has a stoichiometry of four melittin molecules per S100P dimer and is presumably driven by favorable electrostatic interactions between the acidic protein and the basic peptide. In contrast, Ca2+-dependent binding of melittin to S100P occurs with high affinity (Kd approximately 5 microM) has a stoichiometry of two molecules of melittin per S100P dimer, appears to have positive cooperativity, and is driven by hydrophobic interactions. Furthermore, Ca2+-dependent S100P-melittin complex formation is accompanied by significant conformational changes: Melittin, otherwise unstructured in solution, adopts a helical conformation upon interaction with Ca2+-S100P. These results support a model for the Ca2+-dependent conformational switch in S100P for functional target recognition.     

Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus.

The v-erbA oncogene, a transduced copy of a thyroid hormone receptor, plays an important role in establishment of the transformed cell phenotype induced by avian erythroblastosis virus. The ability of thyroid hormone receptors to bind to specific sites on chromatin and to thereby modify the expression of adjacent target genes is a crucial element in their mechanism of action in the normal cell. The v-erbA protein also bound at high affinity to a set of DNA fragments recognized by the rat thyroid hormone receptor, but the relative affinity of the v-erbA protein for the different binding sites was distinct from that previously reported for the thyroid hormone receptors.     

ATP binding to the first nucleotide-binding domain of multidrug resistance protein MRP1 increases binding and hydrolysis of ATP and trapping of ADP at the second domain.

Multidrug resistance protein (MRP1) utilizes two non-equivalent nucleotide-binding domains (NBDs) to bind and hydrolyze ATP. ATP hydrolysis by either one or both NBDs is essential to drive transport of solute. Mutations of either NBD1 or NBD2 reduce solute transport, but do not abolish it completely. How events at these two domains are coordinated during the transport cycle have not been fully elucidated. Earlier reports (Gao, M., Cui, H. R., Loe, D. W., Grant, C. E., Almquist, K. C., Cole, S. P., and Deeley, R. G. (2000) J. Biol. Chem. 275, 13098-13108; Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2000) J. Biol. Chem. 275, 20280-20287) indicate that intact ATP is observed bound at NBD1, whereas trapping of the ATP hydrolysis product, ADP, occurs predominantly at NBD2 and that trapping of ADP at NBD2 enhances ATP binding at NBD1 severalfold. This suggested transmission of a positive allosteric interaction from NBD2 to NBD1. To assess whether ATP binding at NBD1 can enhance the trapping of ADP at NBD2, photoaffinity labeling experiments with [alpha-(32)P]8-N(3)ADP were performed and revealed that when presented with this compound labeling of MRP1 occurred at both NBDs. However, upon addition of ATP, this labeling was enhanced 4-fold mainly at NBD2. Furthermore, the nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), bound preferentially to NBD1, but upon addition of a low concentration of 8-N(3)ATP, the binding at NBD2 increased severalfold. This suggested that the positive allosteric stimulation from NBD1 actually involves an increase in ATP binding at NBD2 and hydrolysis there leading to the trapping of ADP. Mutations of Walker A or B motifs in either NBD greatly reduced their ability to be labeled by [alpha-(32)P]8-N(3)ADP as well as by either [alpha-(32)P]- or [gamma-(32)P]8-N(3)ATP (Hou et al. (2000), see above). These mutations also strongly diminished the enhancement by ATP of [alpha-(32)P]8-N(3)ADP labeling and the transport activity of the protein. Taken together, these results demonstrate directly that events at NBD1 positively influence those at NBD2. The interactions between the two asymmetric NBDs of MRP1 protein may enhance the catalytic efficiency of the MRP1 protein and hence of its ATP-dependent transport of conjugated anions out of cells.     

Structure and assembly of the RNA binding domain of bluetongue virus non-structural protein 2

Bluetongue virus non-structural protein 2 belongs to a class of highly conserved proteins found in orbiviruses of the Reoviridae family. Non-structural protein 2 forms large multimeric complexes and localizes to cytoplasmic inclusions in infected cells. It is able to bind single-stranded RNA non-specifically, and it has been suggested that the protein is involved in the selection and condensation of the Bluetongue virus RNA segments prior to genome encapsidation. We have determined the x-ray structure of the N-terminal domain (sufficient for the RNA binding ability of non-structural protein 2) to 2.4 A resolution using anomalous scattering methods. Crystals of this apparently insoluble domain were obtained by in situ proteolysis of a soluble construct. The asymmetric unit shows two monomers related by non-crystallographic symmetry, with each monomer folded as a beta sandwich with a unique topology. The crystal structure reveals extensive monomer-monomer interactions, which explain the ability of the protein to self-assemble into large homomultimeric complexes. Of the entire surface area of the monomer, one-third is used to create the interfaces of the curved multimeric assembly observed in the x-ray structure. The structure reported here shows how the N-terminal domain would be able to bind single-stranded RNA non-specifically protecting the bound regions in a heterogeneous multimeric but not polymeric complex.     

Rapid evolution of peptide and protein binding properties in vitro.

A significant bottleneck in protein engineering arises from the problem of identifying particular molecules with new functions from a potentially enormous range of peptide or protein variants. Two areas of emerging technology, phage display and multiple peptide synthesis, provide new means of screening huge libraries in vitro for novel binding properties. This review is also published in Current Opinion in Structural Biology 1992, 2:597-604.     

The helicase domain of phage P4 alpha protein overlaps the specific DNA binding domain.

Replication initiation depends on origin recognition, helicase, and primase activities. In phage P4, a second DNA region, the cis replication region (crr), is also required for replication initiation. The multifunctional alpha protein of phage P4, which is essential for DNA replication, combines the three aforementioned activities on a single polypeptide chain. Protein domains responsible for the activities were identified by mutagenesis. We show that mutations of residues G506 and K507 are defective in vivo in phage propagation and in unwinding of a forked helicase substrate. This finding indicates that the proposed P loop is essential for helicase activity. Truncations of gene product alpha (gp alpha) demonstrated that 142 residues of the C terminus are sufficient for specifically binding ori and crr DNA. The minimal binding domain retains gp alpha's ability to induce loop formation between ori and crr. In vitro and in vivo analysis of short C-terminal truncations indicate that the C terminus is needed for helicase activity as well as for specific DNA binding.     

S100P, a novel Ca(2+)-binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca2+ binding properties.

A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca(2+)-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identity with the brain S100 proteins alpha and beta, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/polypeptide chain. The low-affinity site (Kd = 800 microM), which, in analogy to S100 beta, seems to involve the N-terminal EF hand, can be followed by the Ca(2+)-dependent decrease in tyrosine fluorescence. The high-affinity site, provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd = 1.6 microM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionyl-2-dimethylaminonaphthalene (Prodan). The Prodan fluorescence shows a Ca(2+)-dependent red shift of the maximum emission wavelength from 485 nm to 502 nm, which is accompanied by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvent in Ca(2+)-bound S100P, making this region of the molecule, the so-called C-terminal extension, an ideal candidate for a putative Ca(2+)-dependent interaction with a cellular target. In p11, a different member of the S100 family, the C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca(2+)-independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca(2+)-dependent conformational change which involves hydrophobic residues of the C-terminal extension.     

Hepatitis C virus envelope protein E2 does not inhibit PKR by simple competition with autophosphorylation sites in the RNA-binding domain

Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.     

The transforming potential of the c-erbB-2 protein is regulated by its autophosphorylation at the carboxyl-terminal domain.

The mutant c-erbB-2 protein with Glu instead of Val-659 exhibited transforming activity in NIH 3T3 cells. This protein showed enhanced tyrosine kinase activity in vitro and enhanced autophosphorylation at Tyr-1248 located proximal to the carboxyl terminus. Enhanced tyrosine phosphorylation of several cellular proteins was detected in cells expressing the Glu-659 c-erbB-2 protein. Introduction of an additional mutation at the ATP-binding site (Lys-753 to Met) of this protein resulted in abolition of its transforming ability. These data indicate that the transforming potential of c-erbB-2 is closely correlated with elevated tyrosine kinase activity of the gene product. To investigate the role of autophosphorylation in cell transformation, we introduced an additional mutation at the autophosphorylation site of the Glu-659 c-erbB-2 protein (Tyr-1248 to Phe). This mutant protein exhibited lower tyrosine kinase activity and lower transforming activity. On the other hand, when the carboxyl-terminal 230 amino acid residues were deleted from the c-erbB-2 protein, the tyrosine kinase activity and cell-transforming activity of the protein were enhanced. Thus, the carboxyl-terminal domain, which contains the major autophosphorylation site, Tyr-1248, may regulate cellular transformation negatively and autophosphorylation may eliminate this negative regulation.     

Formation of a cruciform structure at the simian virus 40 replication origin abolishes T-antigen binding to the origin in vitro.

Heteroduplex DNA molecules were formed by annealing an intact simian virus replication origin-containing fragment to a mutant derivative lacking the indigenous wild-type 27-base-pair (bp) inverted repeat within this structure and containing a nonhomologous 26-bp inverted repeat sequence in its place. Results of restriction enzyme and S1 endonuclease cleavage analyses strongly suggested that a 13-bp stem-loop structure formed at the site of nonhomology between these two DNAs. This structure lies within the boundary of simian virus 40 T-antigen-binding site 2, and its presence inhibited T-antigen binding to that sequence but not to an adjacent higher-affinity binding site (site 1). Therefore, the conformation of sequences within an otherwise intact T-antigen-binding site can have major effects upon T-antigen binding there.     

The transforming protein of the MC29-related virus CMII is a nuclear DNA-binding protein whereas MH2 codes for a cytoplasmic RNA-DNA binding polyprotein.

The acute avian leukemia viruses MH2 and CMII belong to the group of avian myelocytomatosis viruses, the prototype virus of which is MC29. This group of viruses is characterized by myc-specific oncogenes which are presumably expressed as gag-myc polyproteins. These polyproteins are synthesized in non-producer cells transformed by MH2 and CMII and have mol. wts. of 100 000 (p100) and 90 000 (p90), respectively. Monoclonal antibodies against the N terminus of gag, p19, were used to localize the protein in MH2- and CMII-transformed non-producer fibroblasts. Immunofluorescence and cell fractionation indicated that greater than 90% of p100 from MH2 was located in the cytoplasm, whereas greater than 70% of p90 from CMII resided in the nucleus. Isolation of p100 and p90 by immunoaffinity chromatography resulted in an approximately 2000-fold purification of the two polyproteins. Both of them, as well as p110 of MC29, bound to double-stranded DNA of chick fibroblasts in vitro. However, only the MH2-specific polyprotein p100 bound to RNA in vitro. Such a binding was not observed for p90 or p110, or for the purified gag precursor Pr76. Another polyprotein, gag-erbA, from avian erythroblastosis virus, which is also located in the cytoplasm, did not bind to RNA. Our results indicate that the CMII-specific polyprotein p90 behaved indistinguishably from the p110 of MC29. However, the MH2-specific polyprotein p100 exhibited unique and novel properties which were distinct from a gag-myc-type protein.     

Biochemical and physiological properties of the DNA binding domain of AraC protein

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.     

A tandemly repeated sequence determines the binding domain for an erythrocyte receptor binding protein of P. falciparum

Erythrocyte invasion by the malarial merozoite is a receptor-mediated process, an obligatory step in the development of the parasite. The Plasmodium falciparum protein GBP-130, which binds to the erythrocyte receptor glycophorin, is shown here to encode the binding site in a domain composed of a tandemly repeated 50 amino acid sequence. The amino acid sequence of GBP-130, deduced from the cloned and sequenced gene, reveals that the protein contains 11 highly conserved 50 amino acid repeats and a charged N-terminal region of 225 amino acids. Binding studies on recombinant proteins expressing different numbers of repeats suggest that a correlation exists between glycophorin binding and repeat number. Thus, a repeat domain, a common feature of plasmodial antigens, has been shown to have a function independent of the immune system. This conclusion is further supported by the ability of antibodies directed against the repeat sequence to inhibit the in vitro invasion of erythrocytes by merozoites.