Identification of two separable modules in the duck hepatitis B virus core protein.

Hepadnavirus replication requires the concerted action of the polymerase and core proteins to ensure packaging of the RNA pregenome and DNA maturation. The arginine-rich C terminus of the core protein plays an essential role in both of these steps while being dispensable for nucleocapsid formation. In an attempt to identify other functional domains of the core protein, we performed a series of trans-complementation experiments analyzing the ability of duck and human hepatitis B virus (DHBV and HBV) core protein subunits to support the replication of a core-defective DHBV genome. Plasmids expressing the N-terminal amino acids 1 to 67 or the remaining C-terminal portion, amino acids 67 to 262, of the DHBV core protein were cotransfected into LMH cells along with a replication-deficient construct coding for the DHBV pregenome and polymerase. Neither the N nor the C terminus alone yielded replication-competent core particles. However, cotransfection of plasmids that separately expressed both regions restored a normal replication pattern. Furthermore, the DHBV C terminus but not the N terminus could be replaced by the corresponding domain of the HBV core protein in this assay. Finally, coexpression of the complete HBV core protein and the N terminus from DHBV resulted in DHBV replication, while the HBV core protein alone was not functional. Taken together, these findings suggest a modular organization of the DHBV core protein in which the C terminus is functionally conserved among different hepadnaviruses.     

A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.     

The carboxyl terminus of protein kinase c provides a switch to regulate its interaction with the phosphoinositide-dependent kinase, PDK-1

The function of protein kinase C family members depends on two tightly coupled phosphorylation mechanisms: phosphorylation of the activation loop by the phosphoinositide-dependent kinase, PDK-1, followed by autophosphorylation at two positions in the COOH terminus, the turn motif, and the hydrophobic motif. Here we address the molecular mechanisms underlying the regulation of protein kinase C betaII by PDK-1. Co-immunoprecipitation studies reveal that PDK-1 associates preferentially with its substrate, unphosphorylated protein kinase C, by a direct mechanism. The exposed COOH terminus of protein kinase C provides the primary interaction site for PDK-1, with co-expression of constructs of the carboxyl terminus effectively disrupting the interaction in vivo. Disruption of this interaction promotes the autophosphorylation of protein kinase C, suggesting that the binding of PDK-1 to the carboxyl terminus protects it from autophosphorylation. Studies with constructs of the COOH terminus reveal that the intrinsic affinity of PDK-1 for phosphorylated COOH terminus is over an order of magnitude greater than that for unphosphorylated COOH terminus, contrasting with the finding that PDK-1 does not bind phosphorylated protein kinase C effectively. However, effective binding of the phosphorylated species can be induced by the activated conformation of protein kinase C. This suggests that the carboxyl terminus becomes masked following autophosphorylation, a process that can be reversed by the conformational changes accompanying activation. Our data suggest a model in which PDK-1 provides two points of regulation of protein kinase C: 1) phosphorylation of the activation loop, which is regulated by the intrinsic activity of PDK-1, and 2) phosphorylation of the carboxyl terminus, which is regulated by the release of PDK-1 to allow autophosphorylation.     

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.     

Role of the C terminus in antigen P1 surface localization in Streptococcus mutans and two related cocci.

The C terminus of the major surface protein P1 from Streptococcus mutans is composed of a hydrophilic domain, an LPNTGV motif, a hydrophobic domain, and a charged tail. These features are shared by surface proteins from many gram-positive coccal bacteria. To investigate the role of the C-terminal domains in antigen P1 surface localization, full-length and truncated P1 gene constructs, which were expressed on the shuttle vector pDL276, were transformed into the P1-negative mutant S. mutans SM3352, Streptococcus gordonii DL-1, and Enterococcus faecalis UV202. Transformants were tested for expression of P1 by enzyme-linked immunosorbent assaying and Western blotting. The results showed that full-length P1 was expressed by transformants of all three bacteria and was localized on the cell surface. A fusion protein composed of the Staphylococcus aureus fibronectin binding protein C terminus and the P1 protein N terminus was found to surface localize in S. mutans. Deletion of the entire C-terminal domains resulted in P1 being expressed in the culture supernatant. A P1 truncation, which carried only the hydrophilic domain at its C terminus, was found partially associated with the cell surface. This truncated P1 was readily removed from the isolated cell wall by hot sodium dodecyl sulfate-mercaptoethanol extraction. In contrast, the full-length P1 remained associated with the isolated cell wall after similar treatment, suggesting covalent linkages between the full-length P1 and the cell wall. The results described above showed that antigen P1 was anchored to the cell wall by its C-terminal domains probably via covalent linkages with the cell wall. The results also support a universal mechanism involving the C-terminal domains for protein surface localization among this group of gram-positive bacteria.     

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.     

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin.

Bacillus sphaericus 2362 produces a binary toxin consisting of 51- and 42-kDa proteins, both of which are required for toxicity to mosquito larvae. Upon ingestion by larvae, these proteins are processed to 43 and 39 kDa, respectively. Using site-directed mutagenesis, we have obtained N- and C-terminal deletions of the 51-kDa protein and expressed them in B. subtilis by using the subtilisin promoter. Removal of 21 amino acids from the N terminus and 53 amino acids from the C terminus resulted in a protein with the same electrophoretic properties as the 43-kDa degradation product which accumulates in the guts of mosquito larvae. This protein was toxic only in the presence of the 42-kDa protein. A deletion of 32 amino acids at the N terminus combined with a 53-amino-acid deletion at the C terminus resulted in a protein which retained toxicity. Toxicity was lost upon a further deletion of amino acids at potential chymotrypsin sites (41 at the N terminus, 61 at the C terminus). Comparison of the processing of the 51- and the 42-kDa proteins indicated that in spite of their sequence similarity proteolysis occurred at different sites.     

Characterization of a Matrix Protein in the Gastroliths of the Crayfish Procambarus clarkii

As a first step in understanding the calcification mechanism, a matrix protein in the gastrolith of the crayfish Procambarus clarkii was purified and sequenced. The protein was insoluble in acid, but after trypsin digestion, it dissolved in 6 m urea. The trypsin-digested protein dissolved in urea solution was purified by reversed-phase HPLC and designated gastrolith matrix protein fragment. The fragment had a molecular weight of 9658 and a blocked amino terminus. It had tandemly repeated units not reported before at the central part of the sequence, with each unit being Gly-Ser-X1-X2-Phe as the most typical sequence. This peptide was found associated with chitin, a main component of the organic matrix.     

Peptide vaccine against canine parvovirus: identification of two neutralization subsites in the N terminus of VP2 and optimization of the amino acid sequence.

The N-terminal domain of the major capsid protein VP2 of canine parvovirus was shown to be an excellent target for development of a synthetic peptide vaccine, but detailed information about number of epitopes, optimal length, sequence choice, and site of coupling to the carrier protein was lacking. Therefore, several overlapping peptides based on this N terminus were synthesized to establish conditions for optimal and reproducible induction of neutralizing antibodies in rabbits. The specificity and neutralizing ability of the antibody response for these peptides were determined. Within the N-terminal 23 residues of VP2, two subsites able to induce neutralizing antibodies and which overlapped by only two glycine residues at positions 10 and 11 could be discriminated. The shortest sequence sufficient for neutralization induction was nine residues. Peptides longer than 13 residues consistently induced neutralization, provided that their N termini were located between positions 1 and 11 of VP2. The orientation of the peptides at the carrier protein was also of importance, being more effective when coupled through the N terminus than through the C terminus to keyhole limpet hemocyanin. The results suggest that the presence of amino acid residues 2 to 21 (and probably 3 to 17) of VP2 in a single peptide is preferable for a synthetic peptide vaccine.     

Identification and characterization of the human orthologue of yeast Pex14p

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.     

The N terminus of FliM is essential to promote flagellar rotation in Rhodobacter sphaeroides

FliM is part of the flagellar switch complex. Interaction of this protein with phospho-CheY (CheY-P) through its N terminus constitutes the main information relay point between the chemotactic system and the flagellum. In this work, we evaluated the role of the N terminus of FliM in the swimming behavior of Rhodobacter sphaeroides. Strains expressing the FliM protein with substitutions in residues previously reported in Escherichia coli as being important for interaction with CheY showed an increased stop frequency compared with wild-type cells. In accordance, we observed that R. sphaeroides cells expressing FliM lacking either the first 13 or 20 amino acids from the N terminus showed a stopped phenotype. We show evidence that FliMDelta13 and FliMDelta20 are stable proteins and that cells expressing them allow flagellin export at levels indistinguishable from those detected for the wild-type strain. These results suggest that the N-terminal region of FliM is required to promote swimming in this bacterium. The role of CheY in controlling flagellar rotation in this organism is discussed.     

The functions of the N terminus of the phiX174 internal scaffolding protein, a protein encoded in an overlapping reading frame in a two scaffolding protein system

phiX174 utilizes two scaffolding proteins during morphogenesis, an internal protein (B) and an external protein (D). The B protein induces a conformational change in coat protein pentamers, enabling them to interact with both spike and external scaffolding proteins. While functions of the carboxyl terminus of protein B have been defined, the functions of the amino terminus remain obscure. To investigate the morphogenetic functions of the amino terminus, several 5' deleted genes were constructed and the proteins expressed in vivo. The DeltaNH(2) B proteins were assayed for the ability to complement an ochre B mutant and defects in the morphogenetic pathway were characterized. The results of the biochemical, genetic and second-site genetic analyses indicate that the amino terminus induces conformational changes in the viral coat protein and facilitates minor spike protein incorporation. Defects in conformational switching can be suppressed by substitutions in the external scaffolding protein, suggesting some redundancy of function between the two proteins.     

Peptide-specific antibody for the melibiose carrier of Escherichia coli localizes the carboxyl terminus to the cytoplasmic face of the membrane

The synthetic decapeptide NH2-Cys-Val-Gly-Ala-Val-Ser-Asp-Val-Lys-Ala-COOH (designated MBct10), which corresponds to the carboxyl terminus of the melibiose carrier of Escherichia coli, was synthesized and used to raise antibodies in a rabbit. Anti-MBct10 antibodies recognizes the normal melibiose carrier but not a truncated carrier lacking 14 carboxyl-terminal amino acids. Thus the antibodies are specific for the carboxyl terminus of the carrier and not for other domains of the protein. When right-side-out and inside-out membrane vesicles were probed with anti-MBct10 serum, only the inside-out vesicles bound antibody. The carboxyl terminus of the melibiose carrier protein is therefore exposed on the cytoplasmic surface of the membrane. The co-localization of both NH2- and carboxyl termini to the cytoplasmic surface dictates that the protein cross the membrane an even number of times. These data together with hydrophobicity analysis support a topological model for the melibiose carrier with 10 or 12 transmembrane domains.     

Orientation of the carboxyl terminus of the transposon Tn10-encoded tetracycline resistance protein in Escherichia coli

A site-directed antibody was generated against a synthetic polypeptide corresponding to the 14 amino acid residues of the carboxyl terminus of the Tn10 TetA protein. The antibody reacted preferentially with inside-out vesicles, rather than right-side-out vesicles, prepared from Escherichia coli cells harboring transposon Tn10. When inside-out vesicles were treated with trypsin, the TetA protein was completely digested in the vicinity of the carboxyl terminus, as judged on immunoblot analysis using the antibody. In contrast, when right-side-out vesicles were treated with trypsin, the TetA protein was hardly digested. These results indicate that the carboxyl terminus of TetA is exposed to the cytoplasmic side of the membrane.     

Role of polycationic C-terminal portion in the structure and activity of recombinant human interferon-gamma

Purified recombinant human interferon-gamma, produced in Escherichia coli, was digested with trypsin under mild conditions, resulting in a preparation containing approximately 90% of a Mr = 15,800 protein and 10% of a 14,400 protein. The Mr = 15,800 protein has an intact N terminus and the Mr = 14,400 protein lacks 14 N-terminal residues. Both proteins lack C terminus of approximately 13 residues. This preparation containing the Mr = 15,800 and 14,400 proteins was identical with the intact protein with respect to conformation and dimerization, as analyzed by circular dichroism and gel filtration. However, the antiviral activity of this preparation was 1000-fold lower than that of the intact molecule. Since the majority of this preparation is the Mr = 15,800 protein, these results suggest that the C terminus does not affect the protein conformation and self-association, but greatly alters antiviral activity.     

The COOH terminus of the amelogenin, LRAP, is oriented next to the hydroxyapatite surface

The organic matrix in forming enamel consists largely of the amelogenin protein self-assembled into nanospheres that are necessary to guide the formation of the unusually long and highly ordered hydroxyapatite (HAP) crystallites that constitute enamel. Despite its ability to direct crystal growth, the interaction of the amelogenin protein with HAP is unknown. However, the demonstration of growth restricted to the c-axis suggests a specific protein-crystal interaction, and the charged COOH terminus is often implicated in this function. To elucidate whether the COOH terminus is important in the binding and orientation of amelogenin onto HAP, we have used solid state NMR to determine the orientation of the COOH terminus of an amelogenin splice variant, LRAP (leucine-rich amelogenin protein), which contains the charged COOH terminus of the full protein, on the HAP surface. These experiments demonstrate that the methyl 13C-labeled side chain of Ala46 is 8.0 A from the HAP surface under hydrated conditions, for the protein with and without phosphorylation. The experimental results provide direct evidence orienting the charged COOH-terminal region of the amelogenin protein on the HAP surface, optimized to exert control on developing enamel crystals.     

Protein interactions targeting the latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus to cell chromosomes

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) latent infection depends on the viral episomes in the nucleus being distributed to daughter cells following cell division. The latency-associated nuclear antigen (LANA) is constitutively expressed in all KSHV-infected cells. LANA binds sequences in the terminal repeat regions of the KSHV genome and tethers the viral episomes to chromosomes. To better understand the mechanism of chromosomal tethering, we performed glutathione S-transferase (GST) affinity and yeast two-hybrid assays to identify LANA-interacting proteins with known chromosomal association. Two of the interactors were the methyl CpG binding protein MeCP2 and the 43-kDa protein DEK. The interactions of MeCP2 and DEK with LANA were confirmed by coimmunoprecipitation. The MeCP2-interacting domain was mapped to the previously described chromatin binding site in the N terminus of LANA, while the DEK-interacting domain mapped to LANA amino acids 986 to 1043 in the C terminus. LANA was unable to associate with mouse chromosomes in chromosome spreads of transfected NIH 3T3 cells. However, LANA was capable of targeting to mouse chromosomes in the presence of human MeCP2 or DEK. The data indicate that LANA is tethered to chromosomes through two independent chromatin binding domains that interact with different protein partners.     

An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.