tau Binds and Organizes Escherichia coli Replication Proteins through Distinct Domains

PARTIAL PROTEOLYSIS OF TERMINALLY TAGGED tau  TO DETERMINE CANDIDATE DOMAINS AND TO ASSIGN DOMAIN V AS THE alpha  BINDING DOMAIN*

Dexiang Gao and Charles S. McHenry

From the Department of Biochemistry and Molecular Genetics and Program in Molecular Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The tau  subunit dimerizes Escherichia coli DNA polymerase III core through interactions with the alpha  subunit. In addition to playing critical roles in the structural organization of the holoenzyme, tau  mediates intersubunit communications required for efficient replication fork function. We identified potential structural domains of this multifunctional subunit by limited proteolysis of C-terminal biotin-tagged tau  proteins. The cleavage sites of each of eight different proteases were found to be clustered within four regions of the tau  subunit. The second susceptible region corresponds to the hinge between domain II and III of the highly homologous delta ' subunit, and the third region is near the C-terminal end of the tau -delta ' alignment (Guenther, B., Onrust, R., Sali, A., O'Donnell, M., and Kuriyan, J. (1997) Cell 91, 335-345). We propose a five-domain structure for the tau  protein. Domains I and II are based on the crystallographic structure of delta ' by Guenther and colleagues. Domains III-V are based on our protease cleavage results. Using this information, we expressed biotin-tagged tau  proteins lacking specific protease-resistant domains and analyzed their binding to the alpha  subunit by surface plasmon resonance. Results from these studies indicated that the alpha  binding site of tau  lies within its C-terminal 147 residues (domain V).



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The structurally complex DNA polymerase III holoenzyme is responsible for replication of the majority of the chromosome in Escherichia coli. The polymerase of the enzyme and 3' right-arrow 5' exonuclease proofreading activities are contained within the heterotrimeric DNA polymerase III core (alpha epsilon theta ) subassembly. The holoenzyme contains seven different auxiliary subunits (beta , gamma , delta , delta ', tau , chi , and psi ) that confer a number of special properties requisite for replicative polymerase function (1-4). These properties include a rapid elongation rate, high processivity, and the ability to communicate with primosomal proteins at the replication fork (5-7). The auxiliary subunits are divided between two functional assemblies: a beta 2 sliding clamp processivity factor, and the DnaX complex, a multiprotein ATPase that assembles the beta 2 processivity factor onto the primer-template.

Both the tau  and gamma  subunits of the holoenzyme are expressed from dnaX. Translation of dnaX gene yields the full-length tau  subunit (71 kDa) as well as the gamma  subunit (47 kDa), which corresponds to the N-terminal two-thirds of the tau  sequence (8-11). The gamma  subunit results from -1 translational frameshifting to a frame with an early stop codon. The tau  subunit plays central roles in the structure and function of the holoenzyme. Interactions between the tau  and alpha  subunits result in the formation of a dimeric DNA polymerase III' (alpha epsilon theta )2tau 2 (12, 13). This dimeric polymerase effectively couples synthesis of the leading and lagging strands (12, 14). The tau  subunit binds tightly to alpha , but the shorter dnaX translation product (gamma ) does not. This observation suggested that the C-terminal portion unique to tau  is critical for its interactions with the alpha  subunit. Indeed, the alpha  subunit and C-tau , an OmpT proteolytic fragment corresponding to the 215 C-terminal residues of tau , bind with a 1:1 stoichiometry (15).

Interactions between the tau  subunit and DnaB helicase (DnaB) are critical for rapid movement at the replication fork (16, 17). In systems using the reconstituted DNA polymerase III holoenzyme, tau  subunit-DnaB interactions stimulate the rate of helicase unwinding more than 10-fold to levels approaching the rate of fork progression in vivo. The C-terminal region found in tau  but lacking in gamma  has been implicated in replication fork function. The C-tau fragment was shown to interact with DnaB and to effectively couple the leading strand polymerase with DnaB helicase at the replication fork (15).

The tau  subunits bind gamma delta delta 'chi psi to form the DnaX complex, tau 2gamma 1(delta delta 'chi psi ) (18, 19). The tau  subunit also serves as a bridge between alpha  and a chi -SSB interaction, strengthening the holoenzyme interactions with the single-stranded DNA-binding protein-coated lagging strand at the replication fork (20, 21). As part of the elongation complex, tau  protects beta 2 from removal by exogenous gamma  complex, increasing the processivity of the replicase to the megabase range (22).

Clearly, tau  mediates its functions through interactions with other subunits. To identify distinct structural domains that might mediate these multiple interactions, we performed limited proteolytic digestion of recombinant, biotin-tagged tau . Based on these findings, we constructed plasmids encoding truncated tau  fusion proteins lacking one or more putative structural domains. The relative binding of each resultant purified fusion protein to the alpha  subunit was determined by surface plasmon resonance. These studies enabled the identification of domain V (147 C-terminal amino acid residues) as the alpha  subunit-binding domain of tau .


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains-- E. coli DH5alpha and HB101 were used for initial molecular cloning procedures and plasmid propagation. E. coli BL21 and BL21(lambda DE3) were employed for protein expression.

Chemicals and Reagents-- All proteases were purchased from Roche Molecular Biochemicals or Sigma. d-Biotin was purchased from Sigma. SDS-polyacrylamide gel electrophoresis protein standards were obtained from Amersham Pharmacia Biotech, and prestained molecular mass markers were from Bio-Rad or Life Technologies, Inc. Ni2+-NTA1 resin, QIAquick Gel extraction kits, QIAquick PCR purification kits, and plasmid preparation kits were purchased from Qiagen (Valencia, CA). The Coomassie Plus Protein Assay Reagent and ImmunoPure Streptavidin are vended by Pierce. CM5 sensor chips (research grade), P-20 surfactant, NHS, EDC, and ethanolamine hydrochloride were obtained from BIAcore, Inc. (Piscataway, NJ).

Construction of the Fusion Plasmids-- The N- and C-terminal fusion vectors pPA1-N0 and pPA1-C0 were constructed as previously described (23). The fusion peptides contained a short 13-amino acid biotinylation sequence, a hexahistidine sequence, and a thrombin cleavage site. The induced fusion proteins are under the control of either the T7 promoter of pET-11C vector (Novagen, Madison, WI) or the PA1/04/03 promoter/operator (referred to as PA1). PA1 is a semi-synthetic E. coli RNA polymerase-dependent promoter containing two lac operators (23, 24). The dnaX gene was derived from the pRT610A plasmid in which the dnaX gene was modified at the frameshifting site. This modification results in the specific expression of the tau  subunit; the alternative expression product, gamma , is not encoded by this construct (25).

PA1-N-Delta 1tau plasmid (see Fig. 1A) encodes the tau  protein lacking only the first amino acid (methionine) with an N-terminal fusion peptide placed in frame. PA1-N-Delta 1tau was generated by replacing the alpha -encoding gene dnaE from the vector used as the starting material (plasmid PA1-N0) with dnaX (see Fig. 1A). Oligonucleotides 2782 and 2783 (Table I), which correspond to the codons for tau  amino acids 2-10, were annealed and inserted into pBluescript (KS-) to generate pDG10. The remainder of the dnaX gene sequence was from pDG50, which was derived from pRT610A via elimination of a PstI restriction site. The 2187-base pair NarI/HindIII restriction fragment from pDG50 was ligated to a similarly digested pDG10 vector to generate pDG100. The 2209-base pair PstI/SphI fragment from pDG100 replaced the corresponding fragment in the N-terminal fusion peptide-containing vector pPA1-N0 to yield pPA1-N-Delta 1tau .


                              
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Table I
Oligonucleotides used for construction of tau  deletion fusion proteins

Plasmid PA1-C(0)tau encodes the intact tau  protein tagged with a C-terminal fusion peptide (see Fig. 1B). The NheI/PstI fragment at the C-terminal end of the dnaX sequence within pRT610A was replaced with a PCR-generated fragment to produce pRT610AM, in which the stop codon was replaced with a SpeI cloning site. The 1963-base pair XbaI-SpeI fragment from this plasmid was used to replace the corresponding fragment in the C-terminal fusion peptide-expressing vector pPA1-C0 to produce pPA1-C(0)tau .

PCR was used to generate plasmid PA1-N-Delta 413tau , which lacks the sequences encoding the N-terminal 413 amino acids of tau . Oligonucleotides N-Delta 413P1 and N-Delta 413P2 (Table I) were used with template pPA1-N-Delta 1tau to PCR amplify a truncated dnaX fragment. N-Delta 413P1 contained a PstI site in the noncomplementary 5' region followed by a complementary region extending from codons 414-419. N-Delta 413P2 annealed to a region of dnaX located 100 bases downstream of NheI site. To generate pPA1-N-Delta 413tau , the amplified dnaX fragment was ligated into pPA1-N-Delta 1tau following digestion of the plasmid with PstI and NheI.

Plasmid pET11-N-Delta 496tau lacks the sequences encoding the N-terminal 496 amino acids of tau . Primers N-Delta 496P1 and N-Delta 496P2 were used with template pPA1-N-Delta 1tau to generate a partial dnaX fragment, which contained a PstI site in the noncomplementary 5' region and KpnI restriction site more proximal to the 3' end. The KpnI restriction site was located downstream of the dnaX natural stop codon (see Fig. 1A). This PCR fragment replaced the dnaE gene in PstI/KpnI-digested pET11-N0 (23) to generate pET11-N-Delta 496tau .

Plasmid PA1-C-Delta 147tau lacks the sequences encoding the C-terminal 147 amino acids of tau . PCR primer C-Delta 147P1 annealed to a dnaX sequence located 146 bases upstream of the RsrII cloning site. The other PCR primer C-Delta 147P2 was complementary to the dnaX from codon 494-496, followed by a noncomplementary SpeI cloning site. The two primers were used with template pRT610A to generate a partial dnaX sequence. Following RsrII and SpeI digestion, this fragment replaced the corresponding fragment in pPA1-C(0)tau (see Fig. 1B) to generate pPA1-C-Delta 147tau .

Cell Growth and Induction-- For protein expression applications, E. coli strain BL21 was transformed with plasmids PA1-C(0)tau or PA1-N-Delta 1tau . E. coli strain BL21(lambda DE3) was used for each of the other expression plasmids. E. coli bearing plasmid PA1-N-Delta 1tau was grown at 37 °C to an optical density of 0.8 in 6 liters of F medium (26) containing 100 µg/ml ampicillin. Bacteria transformed with each of the other plasmids were grown to the same density under the same conditions, except that the volume of F medium was 2 liters. The induction process was started by the addition of isopropyl-beta -D-thio-galactoside (final concentration, 1 mM). Additional ampicillin (100 µg/ml) and d-biotin (10 µM) were added to the media at the same time. After 2 h of induction, cells were harvested by centrifugation at 5860 × g for 10 min at 4 °C and resuspended in 1 ml of Tris-sucrose buffer (50 mM Tris-HCl, pH 7.5, and 10% sucrose)/g of cells. Cells were quickly frozen in liquid N2 and stored at -80 °C.

Ni2+-NTA Chromatography-- BL21 cells containing expression plasmids PA1-C(0)tau or PA1-N-Delta 1tau were lysed in the presence of lysozyme (2 mg/g of cells), 2 mM EDTA, 5 mM benzamidine, and 1 mM PMSF for 1 h on ice followed by a 4-min incubation at 37 °C (26). For BL21(lambda DE3) cells containing expression plasmids PA1-C-Delta 147tau , PA1-N-Delta 413tau , or pET11-N-Delta 496tau , the lysis procedure was modified by increasing the concentrations of lysozyme (2.5 mg/g of cells) and EDTA (5 mM) and by extending the heat treatment step to 6 min at 37 °C. Lysates were centrifuged at 23,300 × g at 4 °C for 1 h to remove debris. For purification of N-Delta 1tau , 0.226 g of ammonium sulfate was added to each milliliter of the resulting supernatant and precipitant was collected by centrifugation at 23,300 × g at 4 °C for 1 h. Protein pellets were resuspended to ~30 mg protein/ml in Buffer L (50 mM sodium phosphate, pH 7.6, 500 mM NaCl, 10% glycerol, 0.5 mM PMSF, 0.5 mM benzamidine, and 1 mM imidazole). Ni2+-NTA resin, previously equilibrated with Buffer L, was added to the suspensions for binding. Binding was conducted at 4 °C for 2 h with gentle shaking. Slurries of Ni2+-NTA resin/tau fusion protein complexes were then packed into columns. Columns were washed with 10 column volumes of Buffer L and then with roughly 30 column volumes of Buffer W (50 mM sodium phosphate, pH 7.6, 500 mM NaCl, 20% glycerol, 0.5 mM PMSF, and 0.5 mM benzamidine) plus 23 mM imidazole. Bound N-Delta 1tau protein was eluted with 10 column volumes of a 23-150 mM imidazole gradient in Buffer W. The peak fraction eluted at about 60 mM imidazole. Wash and elution steps were performed at 4 °C.

The purification procedures for C(0)tau , C-Delta 147tau , N-Delta 413tau , and N-Delta 496tau were the same as above, except for modifications of the precipitation, binding, and washing steps for N-Delta 413tau and N-Delta 496tau and a simplified elution step for each of these four proteins. For N-Delta 413tau and N-Delta 496tau , supernatant proteins were precipitated by adding 0.36 g of ammonium sulfate to each milliliter of cell lysates. For these two fusion proteins, the imidazole concentrations in the binding and washing steps were 2 and 15 mM, respectively. The N-Delta 413tau , N-Delta 496tau , C(0)tau , and C-Delta 147tau proteins were each eluted in single steps with 150 mM imidazole in Buffer W.

SDS-Polyacrylamide Gel Electrophoresis-- Proteins were separated by overnight electrophoresis at 65 V on a 10-17.5% SDS-polyacrylamide gradient gel (0.75 × 18 × 16 cm). Gels were stained with a 0.1% solution of Coomassie Brilliant Blue R-250 in 20% methanol and 10% acetic acid and then destained in a solution of 10% methanol and 10% acetic acid.

Biotin Blots-- After separation by SDS-polyacrylamide gel electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 500 mA for 3 h in 25 mM Tris-HCl, 192 mM glycine, pH 8.3, 20% methanol, and 0.01% SDS. Membranes were dipped in methanol and then air-dried for 20 min. Membranes were incubated with alkaline phosphatase-conjugated streptavidin (2 µg/ml) in TBS + 0.05% Tween 20 plus 0.5% nonfat milk for 1 h at room temperature and then washed three times in TBS +0.05% Tween20. Blots were developed in a substrate solution containing nitroblue tetrazolium chloride (0.33 mg/ml) and 5-bromo-4-chloro-3'-indolylphosphate p-toluidine salt (0.165 mg/ml) in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, and 50 mM MgCl2. Reactions were stopped by washing the membranes with distilled water.

Limited Proteolysis-- C(0)tau digestions were carried out in 50 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 10% glycerol, and 0.1 mM EDTA. Proteolytic digestions of C-Delta 213tau were in 50 mM Tris-HCl (pH 7.6), 100 mM NaCl, 5% glycerol, and 10 mM Mg(CH3CO2)2. At different time points, 15-µl aliquots from reaction mixtures were removed, mixed with 8 µl of stop buffer (0.18 M Tris, pH 6.8, 30% sucrose, 6% SDS, 180 mM dithiothreitol), and then immediately boiled for 2 min. Each aliquot contained 3 µg of protein. Digestion products were separated by SDS-polyacrylamide gel electrophoresis and then stained with Coomassie Brilliant Blue or transferred onto a polyvinylidene difluoride membrane for biotin blots.

Protein Sequencing-- After digestion, the selected biotinylated fragments were purified from others by using Ni2+-NTA chromatography with the same buffers used for N-Delta 1tau except that urea was added to the binding and washing buffer at 8 M final concentration. These purified biotinylated fragments were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes in 10 mM CAPS (pH 11.0) and 10% methanol at constant current (0.4 A) for 3 h. The membranes were washed with 20% aqueous methanol and then subjected to N-terminal sequence analysis using standard Edman chemistry (James McManaman, University of Colorado Cancer Center Protein Core Laboratory).

Protein Determinations-- Concentrations of all purified proteins were determined by UV spectroscopy using their extinction coefficients. Concentrations of tau  fusion proteins were determined by the Pierce Coomassie Plus Protein Assay Reagent according to the manufacturer's instructions. Bovine serum albumin (fat-free; Sigma) was used as a standard.

DNA Polymerization Assays-- Activities of tau  fusion proteins were measured by their requirement for reconstitution of holoenzyme activity and by measuring DNA synthesis from a primed M13Gori template (26). Assay mixtures (25 µl) contained 500 pmol of M13Gori (as nucleotide), 165 units (40 ng) of DnaG primase, 1.6 µg of E. coli single-stranded DNA binding protein, 250 fmol each of DNA polymerase III core (alpha epsilon theta ), beta , delta delta ', chi psi , and the test tau  fusion protein (20-50 fmol). Reactions were performed in a buffer containing 50 mM HEPES-KOH (pH 7.5), 10% (v/v) glycerol, 100 mM potassium glutamate, 10 mM dithiothreitol, 10 mM Mg(CH3CO2)2, 200 µg/ml bovine serum albumin, 0.02% (v/v) Tween 20, 48 µM dATP, 48 µM dCTP, 48 µM dGTP, 18 µM [3H]TTP (specific activity, 520 cpm/pmol TTP), and 200 µM rNTP. Assay mixtures were incubated at 30 °C for 5 min, quenched by trichloroacetic acid precipitation, and then filtered through GF/C filters (26). One unit is defined as the amount of enzyme catalyzing the incorporation of 1 pmol of dNTPs/min at 30 °C.

Surface Plasmon Resonance-- A BIAcoreTM instrument was used for protein binding analyses. CM5 research grade sensor chips were used for all experiments. The carboxymethyl dextran matrix of the sensor chip was activated by the NHS/EDC coupling reaction as previously described (27). The matrix was activated using a 220-µl injection of a mixture of 0.2 M EDC and 0.05 M NHS in water to maximize the conversion of the carboxyl groups of the sensor chip matrix to NHS esters. Streptavidin and bovine IgG were sequentially captured onto the matrix by injecting over the chip in 10 mM sodium acetate (pH 4.5) buffer at 0.2 and 0.1 mg/ml, respectively. IgG was used to partially block the negatively charged carboxyl groups on the sensor chip surface. Unreacted NHS ester groups were inactivated using 1 M ethanolamine-HCl (pH 8.5). Typically, 2000 response units (RU) of streptavidin were immobilized. The biotinylated tau  proteins were then injected over the immobilized streptavidin in HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% P-20 surfactant). For kinetic analyses, less than 200 RU of tau  fusion protein were immobilized. Binding studies of tau  to the alpha  subunit (12.5-50 nM) were performed at 20 °C in HKGM buffer (50 mM HEPES, pH 7.4, 100 mM potassium glutamate, 10 mM Mg(CH3CO2)2, and 0.005% P-20 surfactant). A flow rate of 25 µl/min was used for kinetic analyses. Kinetic parameters were determined using the BIAevaluation 2.1 software.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of the C(0)tau and N-Delta 1tau Proteins-- We constructed plasmids PA1-C(0)tau (Fig. 1A) and PA1-N-Delta 1tau (Fig. 1B), which encode C(0)tau and N-Delta 1tau , respectively. Our nomenclature system for truncated fusion proteins indicates the number of terminal residues deleted following the Delta ; the preceding N or C indicates the terminus from which the amino acids were deleted. The fusion peptide is located at the truncated terminus. N-Delta 1tau , for example, indicates that one amino acid was deleted from the N terminus of the tau  sequence and that the fusion peptide was added to the new N terminus. The expressed C(0) tau  and N-Delta 1tau represented ~5% of the total cell protein, as determined by densitometric scans of Coomassie-stained gels.



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Fig. 1.   Construction of plasmids that express tau  with N- or C-terminal tags. The oligonucleotides and PCR primers used are listed in Table I. PA1 and Ptac are the PA1/04/03 and tac promoter, respectively. All of the plasmids shown contain the beta -lactamase gene and lacIq. The fusion peptide region contains a short biotinylation sequence, a hexahistidine sequence, and a thrombin cleavage site (23). A, construction of N-terminal tau  fusion expression plasmid with PA1 promoter. B, construction of C-terminal tau  fusion expression plasmid with PA1 promoter.

C(0)tau and N-Delta 1tau were purified via Ni2+-NTA metal chelating chromatography (Table II). The hexahistidine sequence within the fusion peptide specifically interacts with Ni2+ cheated to the column resin. Lysates (FrI) were prepared from 21 g of C(0)tau or 50 g of N-Delta 1tau expression cells. Both C(0)tau and N-Delta 1tau were recovered at >85% purity after Ni2+-NTA chromatography. The activity peaks of the eluted fractions of both C(0)tau and N-Delta 1tau corresponded to each of their protein peaks (data not shown). Both C(0)tau and N-Delta 1tau were fully active compared with wild-type tau  protein in DNA polymerization assays. C(0)tau and N-Delta 1tau were the only biotinylated proteins in the corresponding eluted fractions examined by the biotin blot analysis (data not shown).


                              
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Table II
Purification of C(0)tau and N-Delta 1tau from overproducing cells

Limited Proteolysis of C(0)tau -- Limited proteolyses were performed to identify protease-sensitive interdomain hinges of the tau  protein. Eight different proteases that encompass a broad spectrum of substrate specificities were tested: chymotrypsin, endoprotease Glu-C (SV8), papain, subtilisin, trypsin, thermolysin, endoprotease Asp-N, and endoprotease Lys-C. We first investigated the effects of varying the protease: C(0)tau ratios and incubation times on the observed proteolytic products. Varying incubation times distinguished the initial cleavage products and also established the differences between stable and unstable fragments. Results from a typical experiment employing chymotrypsin proteolysis are shown in Fig. 2. At short incubation times, 56-, 52-, 48-, and 24-kDa products were observed along with full-length tau  (Fig. 3, lanes 1-3 and 8-10). At longer incubation times, the 56- and 52-kDa products were diminished, whereas the 48- and 24-kDa products and several small bands (<20 kDa) became more intense (Fig. 2, lanes 4-7 and 11-14).



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Fig. 2.   Proteolysis of C(0)tau with chymotrypsin. C(0)tau was subjected to limited proteolysis by two concentrations of chymotrypsin for varying times. Each lane contains 3 µg of C(0)tau protein. After digestion, products were separated by 10-17.5% SDS-polyacrylamide gel and stained with Coomassie Blue. Arrows on the left indicate two examples of cleavage products (48 and 24 kDa) which become more intense with increased digestion time (arrows indicate position in lane1; the same products migrate faster in following lanes because of electrophoresis irregularity). Lane 15, undigested C(0)tau .



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Fig. 3.   Biotin blots of C(0)tau and C-Delta 213tau proteolysis products. After digestion, samples were boiled immediately with the addition of SDS sample buffer, resolved on 10-17.5% SDS-polyacrylamide gel, and subjected to biotin blots as described under "Experimental Procedures." A, C(0)tau was subjected to limited proteolysis with six proteases. Abbreviations used for proteases and their dilution (w/w) and digestion temperature are: C, chymotrypsin, 1:100 at 20 °C; Th, thermolysin, 1:2000 at 37 °C; S, subtilisin, 1:1500 at 20 °C; T, trypsin, 1:2000 at 37 °C; P, papain, 1:500 at 20 °C; SV, endoprotease Glu-C, 1:2000 at 37 °C. Lane 13, C(0)tau , no protease. Arrows on the left indicate the bands (38 kDa, lane 9; 30 kDa, lane 1; 22 kDa, lane1) selected for sequencing. B, C-Delta 213tau was subjected to limited proteolysis with five proteases for several different times. Abbreviations used for proteases and their dilution (w/w) and digestion temperature are: Asp-N, endoprotease Asp-N, 1:500 at 37 °C; Lys-C, endoprotease Lys-C, 1:50 at 37 °C; Glu-C, endoprotease Glu-C, 1:50 at 37 °C; papain, 1:500 at 20 °C; chymtry, chymotrypsin, 1:300 at 20 °C. Arrows on the left indicate the bands (45 kDa, lane 3; 27 kDa, lane 5; 8 kDa, lane 12) selected for sequencing.

Similar experiments were performed for six proteases, and optimal time points were selected for each of them. After separation on SDS-polyacrylamide gels, the digested products were transferred to membranes. Biotin blot analyses were used to identify terminal fragments (Fig. 3A). Several cleavage products resulted from each protease digestion. Several bands with similar mobilities were generated via digestion with different proteases, suggesting that certain regions of C(0)tau were subject to cleavage by multiple proteases. For example, bands of roughly 38 kDa were obtained by digestion with thermolysin, papain, or subtilisin. Bands migrating at ~30 kDa were obtained with either chymotrypsin or subtilisin, and products of about 22 kDa were obtained after digestion with chymotrypsin, SV8, or papain. These observations suggested that C(0)tau contains several protease-sensitive regions.

To facilitate mapping of the cleavage sites closer to the N terminus of tau , C-Delta 213tau , which is equivalent to the gamma  protein plus the fusion peptide at its C terminus, was subjected to limited proteolysis. The conditions for each protease were optimized as described above. Products with apparent molecular masses of 45 kDa were obtained after digestion with endoproteinase Asp-N, endoproteinase Lys-C, or chymotrypsin (Fig. 3B). These and other cleavage products vanished after longer endoproteinase Lys-C digestion because of excessive proteolysis. Intensely staining cleavage products of both 27 and 26 kDa were obtained by digestions with either endoproteinase Asp-N or papain. Products of about 8 kDa were obtained after digestion with either SV8 or chymotrypsin (Fig. 3B).



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Fig. 4.   Protease susceptible sites of the tau  protein. Walker A (GXXXXGKT), zinc module (CX8CXXCXXC), Walker B (DEXX), and SRC motifs are described (30). A total of 11 cleavage sites were obtained by N-terminal amino acid sequencing of the selected biotinylated proteolytic products. The lower panel shows potential structural domains of the tau  protein. Domain I, 1-179 amino acids; domain II, 180-221 amino acids; domain III, 229-382 amino acids; domain IV, 413-496 amino acids; and domain V, 497-643 amino acids.

Seven biotinylated fragments were selected for N-terminal amino acid sequencing analysis for the identification of their cleavage sites. The 22- and 30-kDa chymotryptic fragments were chosen for analysis, as was the 38-kDa fragment resulting from digestion with papain (Fig. 3A). Several products obtained from some of the more specific proteases were also subjected to sequence analyses. These included the 27- and 45-kDa products generated via endoproteinase Asp-N treatment, as well as an 8-kDa product from the SV8 digestion (Fig. 3B). Further, a 20-kDa cleavage product generated by chymotrypsin digestion after prolonged incubation time (data not shown) was also evaluated. Eight corresponding cleavage sites were identified (Fig. 4). Cleavage sites N-terminal to amino acid residues 106 (Asp) and 109 (Asp) corresponded to endoproteinase Asp-N digestion products that migrated at about 45 kDa; residue 222 (Asp) was the proteolytic site resulting in the 27-kDa fragment (Fig. 3B). Papain cleaved between residues 382 (Ala) and 383 (Val) to generate the 38-kDa fragment (Fig. 3A). The 8-kDa fragment generated by SV8 (Fig. 3B) was due to cleavage between residues 407 (Glu) and 408 (Thr). The 30- and 22-kDa chymotryptic fragments (Fig. 3A) resulted from cleavage C-terminal to residues 413 (Leu) and 478 (Trp), respectively. The 20-kDa chymotryptic fragment was due to cleavage after residue 496 (Ala). These cleavage sites are consistent with the substrate specificities of the respective proteases used.



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Fig. 5.   Purified truncated tau  fusion proteins. The upper panel shows the truncated fusion proteins of tau  used in BIAcore analysis. Both C-Delta 147tau and N-Delta 496tau are constructed based on tau  proteolytic site at amino acid 496 and contain domains I-IV and domain V, respectively. N-Delta 413tau is based on tau  proteolytic site at amino acid 413 and contains domain IV+V. The rectangular box represents the fusion peptide. The lower panel is the Coomassie Blue-stained 12.5% SDS-polyacrylamide gel of the above proteins after Ni2+-NTA chromatography with each lane containing approx. 1.5 µg of protein. Lane 1, C(0)tau . Lane 2, C-Delta 147tau . Lane 3, N-Delta 413tau (upper band). Lane 4, N-Delta 496tau .

The probable cleavage site resulting in the 26-kDa endoproteinase Asp-N restriction product was deduced based on that the substrate specificity of that protease and the distribution of aspartate residues in the sequence of tau . Taking an experimental error of ±10% for molecular mass determination via SDS-polyacrylamide gel electrophoresis into account, the cleavage site resulting in the 27-kDa fragment would be predicted to be roughly 9 residues C-terminal to that of the 26-kDa fragment (Asp222). There are two aspartates C-terminal to Asp222: those at positions 229 and 245. Thus, the 26-kDa endoproteinase Asp-N fragment is likely due to cleavage N-terminal to Asp229. Similarly, in consideration of the substrate preferences of papain in light of the known cleavage site of the 27-kDa endoproteinase Asp-N product, it seems likely that the 26-kDa papain digestion product is due to cleavage of bond(s) involving Gly228 and/or Gly230. These probable cleavage sites (228, 229, and 230) together with the eight cleavage sites determined by sequence analysis all cluster at four regions of the tau  subunit: amino acid residues 106-109, 222- 230, 383-413, and 478-496.

The N-terminal half of tau  shares high sequence similarity with delta ' (28-30), another component of the DnaX complex. The crystal structure of delta ' contains three domains, and sequence alignment predicts a similar three-domain structure for the N-terminal half of tau  (30). The majority of the cleavage sites we observed agree and extend this prediction to a five domain model for tau  (see "Discussion" for detail): amino acid residues 1-179 as domain I, 180-221 as domain II, 230-382 as domain III, 413-496 as domain IV, and 497-643 as domain V (Fig. 4).

Expression and Purification of Biotin and Hexahistidine-tagged DnaX-- C-tau , the unique C-terminal portion of tau , is required for binding to alpha  (15). C-tau contains the majority of predicted domain IV and the entire domain V. The fusion protein N-Delta 413tau (domains IV and V) was expressed and purified so that it could be used as a tool for mapping the alpha  binding domain of the tau  subunit. We also expressed fusion proteins corresponding to domain V by itself (N-Delta 496tau ), and domains I-IV (C-Delta 147tau ).

The expression levels of C-Delta 147tau , N-Delta 413tau , and N-Delta 496tau were ~3, 1, and 5% of total cell protein, respectively. All three of these fusion proteins were soluble. After purification by Ni2+-NTA chromatography, C-Delta 147tau and N-Delta 496tau were obtained at greater than 80% purity, and N-Delta 413tau was obtained at over 65% purity as determined by scanning densitometry (Fig. 5). 11.5 mg of C-Delta 147tau , 8 mg of N-Delta 496tau , and 5.3 mg of N-Delta 413tau were purified from 1250, 330, and 1200 mg of total protein from cell lysates, respectively. C-Delta 147tau , N-Delta 413tau , and N-Delta 496tau were the only biotinylated proteins in the corresponding eluted fractions examined by the biotin blot analysis. Thus, the biotinylated tau  fusion proteins were presumed to be the only proteins captured onto the BIAcore sensor chip during the immobilization step.2 C-Delta 147tau is as active as C(0)tau in DNA polymerization assays.

The C-terminal Domain of tau  Binds to alpha -- The interaction between alpha  and C(0)tau was first assessed using BIAcore methodology. Streptavidin was chemically coupled to the CM5 sensor chip, and C(0)tau was immobilized via biotin-streptavidin interaction. Dilutions of alpha  were injected over and bound the immobilized C(0)tau (Fig. 6A). The off rate (koff) was determined after saturating tau  with alpha  to eliminate artifacts arising from re-association. The calculated Kd was ~4 nM (Table III).



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Fig. 6.   BIAcore analysis of the interaction of alpha  with immobilized C(0)tau , N-Delta 413tau , N-Delta 496tau , C-Delta 147tau , and N-Delta 1tau . Five biotinylated tau -fusion proteins were captured onto the streptavidin chips as described under "Experimental Procedures." A streptavidin derivatized flow cell lacking bound tau  protein provided the blank control, which was subtracted from the data shown. The analysis of the interaction with alpha  was performed in HKGM buffer. A-C, sensorgrams of overlays of the indicated concentrations of alpha  injected over the immobilized C(0)tau (150 RU), N-Delta 413tau (710 RU), and N-Delta 496tau (34 RU) flow cells for 3 min at 25 µl/min, respectively. D, 720 RU of C(0)tau and 750 RU of C-Delta 147tau were captured onto streptavidin flow cells, respectively. 50 nM of alpha  was injected over each of these two and the control flow cells in HKGM buffer at 2 µl/min for 15 min. E, 150 RU of N-Delta 1tau were captured onto streptavidin flow cell. 20 nM of alpha  was injected over the flow cell in HKGM buffer at 25 µl/min for 4 min.


                              
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Table III
BIAcore analyses of the binding of alpha  to tau  derivatives

The interaction between alpha  and N-Delta 413tau was characterized by an association rate similar to that observed for C(0)tau -alpha binding; however, its dissociation rate was at least 1000-fold slower (Fig. 6B). This indicated that residues sufficient for specific binding to the alpha  subunit lie within the C-terminal 230 amino acid residues of tau . C-Delta 147tau (domains I-IV) and N-Delta 496tau (domain V) were then immobilized on the streptavidin chips separately to further limit the alpha -binding region. Binding to the alpha  subunit was observed with N-Delta 496tau but not C-Delta 147tau (Fig. 6, C and D), strongly suggesting that domain V functions as the alpha -binding component of the tau  subunit. The off rates (koff ) of both alpha -N-Delta 496tau and alpha -N-Delta 413tau interactions were extremely slow (Fig. 6, B and C). Within the 30-min dissociation period, the response unit changes were within the machine noise level (10 RU), precluding the calculation of a dissociation rate constant, but permitting limits to be placed on the off rates and the III-V domains (Table III).

The interaction between alpha  and N-Delta 1tau was examined to investigate whether the weaker interaction between C(0)tau -alpha might be due to interference by the proximity of the peptide tag component of C(0)tau to the alpha -binding domain. The N-Delta 1tau -alpha interaction was characterized by a dissociation rate similar to that observed for the N-Delta 496tau -alpha interaction but much slower than that detected for the C(0)tau -alpha interaction (Fig. 6E). This suggests that the C-terminal tag interferes with the tau -alpha interaction. We also examined the C(0)tau -alpha interaction in the presence of the auxiliary subunit delta , delta ', and chi psi and ATP. The presence of delta , delta ', and chi psi and ATP did not make obvious changes in the rate of C(0)tau -alpha interaction, suggesting that the delta delta 'chi psi -tau interaction and alpha -tau interaction are independent events.

The calculated stoichiometry of N-Delta 496tau :alpha (1:0.65) was similar to that obtained for the full-length tau  standards (1:0.75 and 1:0.65 for N-Delta 1tau and C(0)tau , respectively). This indicates that domain V, expressed alone, is properly folded and functional.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We report here an effective approach to mapping heterologous protein-protein interacting domains. We improved upon our previously reported mapping method (23) by combining it with limited proteolysis to more precisely identify probable interdomain linkers. This modification improves the probability that the expressed proteins will contain intact domains. Using C-terminal biotin-tagged proteins in our limited proteolysis approach allowed us to distinguish terminal fragments from internal fragments by using streptavidin-conjugated detection reagents. The biotin tag also enabled rapid purification of the terminal fragment for sequence analysis and precise localization of the cleavage sites.

We chose eight proteases with different substrate specificities to identify potential interdomain hinges. As exposed sequences, interdomain hinges are generally susceptible to cleavage by more than one protease. Digestion was monitored as a function of time to help distinguish relatively stable products. Cleavage products common to more than one protease were selected for identification of the precise sites of cleavage. Eleven cleavage sites were obtained following N-terminal amino acid sequencing of the selected proteolysis products. The cleavage sites were clustered within four regions: 106-109, 222-230, 383-413, and 478-496.

The delta ' subunit shares a 34% sequence similarity with the N-terminal region of both tau  and gamma  and aligns to the N-terminal 370-amino acid sequence of DnaX proteins (28, 30), which corresponds to over half of the tau  sequence. There are several known motifs conserved between these two proteins, including the Walker A (GXXXXGKT),3 Walker B (DEXX), zinc module (CX8CXXCXXC), and SRC sequences (Fig. 4) (30). The delta ' crystal structure showed three domains in a C-shaped configuration, suggesting that the N-terminal half of tau  also contains three domains, based on their sequence homology (30). Their alignment suggested that tau  amino acid residues 1-179 were domain I and that residues180-221 were domain II. Less certain was the assignment of domain III to the residues following 226. Our results support the prediction that the N-terminal end of tau  and delta ' have similar structures. One of the tau  protein cleavage sites (Asp222-Gln223) is located within the stretch predicted to be a hinge between domains II and III. The following cleavage site (Ala382-Val383) occurs just 13 residues C-terminal to the end of the tau -delta ' alignment and may define the C-terminal boundary of the third domain of tau . We observed no cleavage between domains I and II, suggesting that this hinge was not accessible for proteolysis under the conditions employed in these studies. Based on the most N-terminal cleavage site identified (Arg105-Asp106), we constructed a plasmid encoding tau  amino acid residues 1-105. The resultant protein was not stable in expression cells (data not shown). Both the residue Asp106 of tau  and the corresponding residue Glu95 of delta ' are predicted to be in a similar position of a helical region revealed by the PHD program (data not shown). The residue Glu95 is on the surface of domain I of delta ' (30). Above results suggest that tau  residues 1-105 do not form an intact structural domain by themselves.

Two cleavage sites (Glu407-Thr408 and Leu413-Ala414) occur C-terminal to Ala382-Val383. These cleavage sites were identified via sequence analyses of different proteolytic fragments (Fig. 3A). 30 residues lie between the scissile bonds at Val383 and Leu413. The presence of at least three cleavage sites within this 30-amino acid stretch suggests that it is highly exposed to proteases and may exist as a nonstructured region of the protein. Therefore, no domain was assigned to the stretch flanked by residues 383 and 413. This analysis does not preclude the formation of structures resulting from association with other molecules not present in our proteolysis experiments domains IV and V were assigned to the remaining sequence at the C terminus of tau . Domain IV is composed of 17 amino acid residues in common with the gamma  translation product and 66 residues unique to the C-terminal sequence of the tau . Domain V corresponds to 147 amino acid residues of the C-terminal end of tau . alpha  interacts with the C-terminal region unique to tau  containing domains IV and V. To further limit the alpha  binding domain of tau , BIAcore analysis was performed. Domain V alone bound alpha  with domains III-V in the pM range. The interaction between alpha  and N-Delta 1tau was of a similar magnitude, indicating that domain V contains all of the binding energy for alpha -tau interaction. This also suggests that our domain assignments for the C-terminal sequence of tau  accurately reflect both the structural and functional integrity of domain V. C-terminal tagged tau  bound alpha  with 1000-fold lower affinity, whereas tagging at the N terminus did not reduce alpha  binding activity in the BIAcore assay. This observation is consistent with localization of the tau  binding sites for alpha  at or near the C terminus of domain V.

Based on our proteolysis data and sequence alignment with the homologous protein delta ', considered in light of the delta ' crystal structure, we assigned five structural domains to the tau  subunit. Final proof of the domain assignments awaits more complete determination of the structure of the tau -subunit.


    ACKNOWLEDGEMENT

We thank Dr. John Kuriyan for sharing the delta ' structure and coordinates with us in advance of publication and Dr. Deborah Wilkinson-Fitzgerald for writing and editorial assistance on this and the following two papers.


    FOOTNOTES

* This work was supported by Grant GM35695 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M009828200

2 A portion of the same preparation of N-Delta 413tau used in BIAcore experiments was chromatographed over a soft link, soft release monomeric avidin column (Promega) equilibrated in Buffer containing 50 mM HEPES (pH 7.4), 100 mM sodium glutamate, 5% (v/v) glycerol, and 0.5 mM PMSF. The column was washed with five column volumes of the equilibration buffer. The bound protein was eluted from the column with 5 mM biotin in equilibration buffer. The N-Delta 413tau band was the only band detected in a Coomassie Blue-stained SDS-polyacrylamide gel electrophoresis of the eluted fractions.

3 delta ' sequence has a clearly derivative but mutated Walker A motif so it cannot bind ATP.


    ABBREVIATIONS

The abbreviations used are: NTA, nitrilotriacetic acid; CAPS, 3-cyclohexylamino-1-propanesulfonic acid; PCR, polymerase chain reaction; NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-[(3-dimethylamino)propyl]-carbodiimide; PMSF, phenylmethylsulfonyl fluoride; RU, response units.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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