tau binds and organizes Escherichia coli replication proteins through distinct domains: domain III, shared by gamma and tau, oligomerizes DnaX.

The tau and gamma proteins of the DNA polymerase III holoenzyme DnaX complex are products of the dnaX gene with gamma being a truncated version of tau arising from ribosomal frameshifting. tau is comprised of five structural domains, the first three of which are shared by gamma (Gao, D., and McHenry, C. (2001) J. Biol. Chem. 276, 4433-4453). In the absence of the other holoenzyme subunits, DnaX exists as a tetramer. Association of delta, delta', chi, and psi with domain III of DnaX(4) results in a DnaX complex with a stoichiometry of DnaX(3)deltadelta'chipsi. To identify which domain facilitates DnaX self-association, we examined the properties of purified biotin-tagged DnaX fusion proteins containing domains I-II or III-V. Unlike domain I-II, treatment of domain III-V, gamma, and tau with the chemical cross-linking reagent BS3 resulted in the appearance of high molecular weight intramolecular cross-linked protein. Gel filtration of domains I-II and III-V demonstrated that domain I-II was monomeric, and domain III-V was an oligomer. Biotin-tagged domain III-V, and not domain I-II, was able to form a mixed DnaX complex by recruiting tau, delta, delta', chi, and psi onto streptavidin-agarose beads. Thus, domain III not only contains the delta, delta', chi, and psi binding interface, but also the region that enables DnaX to oligomerize.     

Tau binds and organizes Escherichia coli replication proteins through distinct domains. Domain III, shared by gamma and tau, binds delta delta ' and chi psi

The DnaX complex of the DNA polymerase holoenzyme assembles the beta(2) processivity factor onto the primed template enabling highly processive replication. The key ATPases within this complex are tau and gamma, alternative frameshift products of the dnaX gene. Of the five domains of tau, I-III are shared with gamma In vivo, gamma binds the auxiliary subunits deltadelta' and chipsi (Glover, B. P., and McHenry, C. S. (2000) J. Biol. Chem. 275, 3017-3020). To localize deltadelta' and chipsi binding domains within gamma domains I-III, we measured the binding of purified biotin-tagged DnaX proteins lacking specific domains to deltadelta' and chipsi by surface plasmon resonance. Fusion proteins containing either DnaX domains I-III or domains III-V bound deltadelta' and chipsi subunits. A DnaX protein only containing domains I and II did not bind deltadelta' or chipsi. The binding affinity of chipsi for DnaX domains I-III and domains III-V was the same as that of chipsi for full-length tau, indicating that domain III contained all structural elements required for chipsi binding. Domain III of tau also contained deltadelta' binding sites, although the interaction between deltadelta' and domains III-V of tau was 10-fold weaker than the interaction between deltadelta' and full length tau. The presence of both delta and chipsi strengthened the delta'-C(0)tau interaction by at least 15-fold. Domain III was the only domain common to all of tau fusion proteins whose interaction with delta' was enhanced in the presence of delta and chipsi. Thus, domain III of the DnaX proteins not only contains the deltadelta' and chipsi binding sites but also contains the elements required for the positive cooperative assembly of the DnaX complex.     

Carboxyl-terminal domain III of the delta' subunit of the DNA polymerase III holoenzyme binds delta

The delta and delta' subunits are essential components of the DNA polymerase III holoenzyme, required for assembly and function of the DnaX-complex clamp loader (tau2gammadeltadelta'chipsi). The x-ray crystal structure of delta' contains three structural domains (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localize the delta-binding domain of delta' to a carboxyl-terminal domain III by quantifying the interaction of delta with a series of delta' fusion proteins lacking specific domains. Purification and immobilization of the fusion proteins were facilitated by the inclusion of a tag containing hexahistidine and a short biotinylation sequence. Both NH2- and COOH-terminal-tagged full-length delta' were soluble and had specific activities comparable with that of native delta'. delta and delta' form a 1:1 heterodimer with a dissociation constant (K(D)) of 5 x 10(-7) m determined by equilibrium sedimentation. The K(D) determined by surface plasmon resonance was comparable. Domain III alone bound delta at an affinity comparable to that of wild type delta', whereas proteins lacking domain III did not bind delta. Using a panel of domain-specific anti-delta' monoclonal antibodies, we found that two of the domain III-specific monoclonal antibodies interfered with delta-delta' interaction and abolished the replication activity of DNA polymerase-III holoenzyme.     

Carboxyl-terminal domain III of the delta' subunit of DNA polymerase III holoenzyme binds DnaX and supports cooperative DnaX complex assembly

The delta' subunit of the DNA polymerase-III holoenzyme is a key component of the DnaX complex; it is required for loading the beta(2) processivity factor onto a primed template. The x-ray crystal structure of delta' indicates a three domain C-shaped structure (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). In this study, we localized the DnaX-binding domain of delta' to its carboxyl-terminal domain III by quantifying protein-protein interactions using a series of delta' fusion proteins lacking specific domains. The fusion protein corresponding to domain III of delta' bound to DnaX with an affinity approaching that of full-length delta'. In contrast, a construct bearing delta' domains I-II did not bind DnaX at detectable levels. The presence of delta and chi psi strengthened the interaction of DnaX with full-length delta' and delta' domain III. Thus, domain III of delta' not only contains the DnaX-binding site, but also contains the elements required for positive cooperative assembly of the DnaX complex. A domain III-specific anti-delta' monoclonal antibody interfered with DnaX complex formation and abolished the replication activity of DNA polymerase III holoenzyme.     

Assembly of DNA polymerase III holoenzyme: co-assembly of gamma and tau is inhibited by DnaX complex accessory proteins but stimulated by DNA polymerase III core.

Although the two alternative Escherichia coli dnaX gene products, tau and gamma, are found co-assembled in purified DNA polymerase III holoenzyme, the pathway of assembly is not well understood. When the 10 subunits of holoenzyme are simultaneously mixed, they rapidly form a nine-subunit assembly containing tau but not gamma. We developed a new assay based on the binding of complexes containing biotin-tagged tau to streptavidin-coated agarose beads to investigate the effects of various DNA polymerase III holoenzyme subunits on the kinetics of co-assembly of gamma and tau into the same complex. Auxiliary proteins in combination with delta' almost completely blocked co-assembly, whereas chipsi or delta' alone slowed the association only moderately compared with the interaction of tau with gamma alone. In contrast, DNA polymerase III core, in the absence of deltadelta' and chipsi, accelerated the co-assembly of tau and gamma, suggesting a role for DNA polymerase III' [tau(2)(pol III core)(2)] in the assembly pathway of holoenzyme.     

Structure-function relationships of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi

Eukaryotic and archaeal initiation factor 2 (e- and aIF2, respectively) are heterotrimeric proteins (alphabetagamma) supplying the small subunit of the ribosome with methionylated initiator tRNA. The gamma subunit forms the core of the heterotrimer. It resembles elongation factor EF1-A and ensures interaction with Met-tRNA(i)(Met). In the presence of the alpha subunit, which is composed of three domains, the gamma subunit expresses full tRNA binding capacity. This study reports the crystallographic structure of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi and that of a derived C-terminal fragment containing domains 2 and 3. The obtained structures are compared with those of N-terminal domains 1 and 2 of yeast and human eIF2alpha and with the recently determined NMR structure of human eIF2alpha. We show that the three-domain organization in the alpha subunit is conserved in archaea and eukarya. Domains 1 and 2 form a rigid body linked to a mobile third domain. Sequence comparisons establish that the most conserved regions in the aIF2alpha polypeptide lie at opposite sides of the protein, within domain 1 and domain 3, respectively. These two domains are known to exhibit RNA binding capacities. We propose that domain 3, which is known to glue the alpha subunit onto the gamma subunit, participates in Met-tRNA(i)(Met) binding while domain 1 recognizes either rRNA or mRNA on the ribosome. Thereby, the observed structural mobility within the e- and aIF2alpha molecules would be an integral part of the biological function of this subunit in the heterotrimeric e- and aIF2alphabetagamma factors.     

A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau

The protein domain responsible for the interaction of tau with tubulin has been identified. Biophysical studies indicated that the synthetic peptide Val187-Gly204 (VRSKIG-STENLKHQPGGG) from the repetitive sequence on tau binds to two sites on the tubulin heterodimer and to one site on each of the microtubule-associated protein-interacting C-terminal tubulin peptides alpha(430-441) and beta(422-434). The binding data showed a relatively stronger interaction of Val187-Gly204 with beta(422-434) as compared to that with alpha(430-441). The interaction of this tau peptide with either alpha or beta tubulin peptides appears to be associated with conformational changes in both the tau and the tubulin peptides. The beta tubulin peptide also appears to induce a structural change of tau fragment Val218-Gly235. Interestingly, tau peptides Val187-Gly204 and Val218-Gly235 induced tubulin self-assembly in a cold-reversible fashion, and incorporated into the assembled polymers. The specificity of the interaction of the tau peptide was supported by the competition of tau protein for the interaction with the tubulin polymer. In addition, the tau peptide appears to contain the principal antigenic determinant(s) recognized by anti-idiotypic antibodies that react with the tubulin binding domains on microtubule-associated proteins. The present findings together with the demonstration of the presence of multiple sites for the binding of the alpha(430-441) and beta(422-434) tubulin fragments to tau, and the existence of repetitive sequences on tau, strongly support the hypothesis that the region of tau defined by the repetitive sequences is involved in its interaction with tubulin.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

A three-domain structure for the delta subunit of the DNA polymerase III holoenzyme delta domain III binds delta' and assembles into the DnaX complex.

Using psi-BLAST, we have developed a method for identifying the poorly conserved delta subunit of the DNA polymerase III holoenzyme from all sequenced bacteria. This approach, starting with Escherichia coli delta, leads not only to the identification of delta but also to the DnaX and delta' subunits of the DnaX complex and other AAA(+)-class ATPases. This suggests that, although not an ATPase, delta is related structurally to the other subunits of the DnaX complex that loads the beta sliding clamp processivity factor onto DNA. To test this prediction, we aligned delta sequences with those of delta' and, using the start of delta' Domain III established from its x-ray crystal structure, predicted the juncture between Domains II and III of delta. This putative delta Domain III could be expressed to high levels, consistent with the prediction that it folds independently. delta Domain III, like Domain III of DnaX and delta', assembles by itself into a complex with the other DnaX complex components. Cross-linking studies indicated a contact of delta with the DnaX subunits. These observations are consistent with a model where two tau subunits and one each of the gamma, delta', and delta subunits mutually interact to form a pentameric functional core for the DnaX complex.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

The DnaX-binding subunits delta' and psi are bound to gamma and not tau in the DNA polymerase III holoenzyme

The DnaX complex subassembly of the DNA polymerase III holoenzyme is comprised of the DnaX proteins tau and gamma and the auxiliary subunits delta, delta', chi, and psi, which together load the beta processivity factor onto primed DNA in an ATP-dependent reaction. delta' and psi bind directly to DnaX whereas delta and chi bind to delta' and psi, respectively (Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13348-13357). Until now, it has been unclear which DnaX protein, tau or gamma, in holoenzyme binds the auxiliary subunits delta, delta', chi,and psi. Treatment of purified holoenzyme with the homobifunctional cross-linker bis(sulfosuccinimidyl)suberate produces covalently cross-linked gamma-delta' and gamma-psi complexes identified by Western blot analysis. Immunodetection of cross-linked species with anti-delta' and anti-psi antibodies revealed that no tau-delta' or tau-psi cross-links had formed, suggesting that the delta' and psi subunits reside only on gamma within holoenzyme.     

Two functional domains of the epsilon subunit of DNA polymerase III

The epsilon subunit is the 3'-->5' proofreading exonuclease that associates with the alpha and theta subunits in the E. coli DNA polymerase III. Two fragments of the epsilon protein were prepared, and binding of these epsilon fragments with alpha and theta was investigated using gel filtration chromatography and exonuclease stimulation assays. The N-terminal fragment of epsilon, containing amino acids 2-186 (epsilon186), is a relatively protease-resistant core domain of the exonuclease. The purified recombinant epsilon186 protein catalyzes the cleavage of 3' terminal nucleotides, demonstrating that the exonuclease domain of epsilon is present in the N-terminal region of the protein. The absence of the C-terminal 57 amino acids of epsilon in the epsilon186 protein reduces the binding affinity of epsilon186 for alpha by at least 400-fold relative to the binding affinity of epsilon for alpha. In addition, stimulation of the epsilon186 exonuclease by alpha using a partial duplex DNA is about 50-fold lower than stimulation of the epsilon exonuclease by alpha. These results indicate that the C-terminal region of epsilon is required in the epsilonalpha association. To directly demonstrate that the C-terminal region of epsilon contains the alpha-association domain fusion protein, constructs containing the maltose-binding protein (MBP) and fragments of the C-terminal region of epsilon were prepared. Gel filtration analysis demonstrates that the alpha-association domain of epsilon is contained within the C-terminal 40 amino acids of epsilon. Also, the epsilon186 protein forms a tight complex with theta, demonstrating that the association of theta with epsilon is localized to the N-terminal region of epsilon. Association of epsilon186 and theta is further supported by the stimulation of the epsilon186 exonuclease in the presence of theta. These data support the concept that epsilon contains a catalytic domain located within the N-terminal region and an alpha-association domain located within the C-terminal region of the protein.     

Common antigenic determinants of the tubulin binding domains of the microtubule-associated proteins MAP-2 and tau.

The structural-functional aspects of the tubulin binding domain on the microtubule-associated protein MAP-2, and its relationship with the tubulin binding domain on tau, were studied using anti-idiotypic antibodies that react specifically with the epitope(s) on MAPs involved in their interaction with tubulin in addition to other tau and MAP-2 specific antibodies. Previous studies showed that MAP-2 and tau share common binding sites on tubulin defined by the peptide sequences alpha (430-441) and beta (422-434) of tubulin subunits. Furthermore, binding experiments revealed the existence of multiple sites for the interaction of the alpha- and beta-tubulin peptides with MAP-2 and tau. Most recent studies showed that the synthetic tau peptide Val187-Gly204 (VRSKIGSTENLKHQPGGG) from the repetitive sequence on tau defines a tubulin binding site on tau. Our present immunological studies using anti-idiotypic antibodies which interact with the synthetic tau peptide and antibodies against the Val187-Gly204 tau peptide indicate that MAP-2 and tau share common antigenic determinants at the level of their respective tubulin binding domains. These antigenic determinants appear to be present in the 35 kDa tubulin binding fragment of MAP-2 and in 18-20 kDa chymotryptic fragments containing the tubulin binding site(s) on MAP-2. These findings, along with structural information on these proteins, provide strong evidence in favor of the hypothesis that tubulin binding domains on MAP-2 and tau share similar structural features.     

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)