Site-directed mutagenesis of the GTP-binding domain of β-tubulin

J. Mol. Biol. (1992) 227, 307-321

Site-directed Mutagenesis of the GTP-binding Domain of p-Tubulin George W. Farr and Himan

Sternlichtt

Department of Pharmacology Case Western Reserve University Cleveland, OH 44106, U.S.A. (Received 14 June 1991; accepted 8 May 1992) Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric with fl-tubulin and binds effector of microtubule assembly, requires Mg ‘+ for its interaction as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The st~ructural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of p-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of B-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. Gne mutation at Asp224 reduced the affinity of P-tubulin for GTP in the presence but not in t,he on nucleotide binding is consistent with absence of Mg ‘+. The specific effect of this mutation an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins. Keywords:

nucleotide-binding;

protein structure; site-directed magnesium; microtubule-assembly

in vitro translation;

I= Introduction

The mechanism by which GTP and GDP produce their opposing effects is not understood. Previous studies have largely focused on the biochemical aspects of nucleotide binding, namely the role of GTP hydrolysis in microtubule assembly and dynamics (Carlier et al, 1989; Stewart et al., 1990; Caplow & Shanks, 1990). While these studies have done much to clarify the underlying molecular mechanisms, the structural basis for the competing effects of GTP and GDP remains obscure. GTP binding occurs as the MgGTP complex and requires the Mg2+ moiety. In contrast, GDP binding is independent of Mg ‘+ (Huang et al., 1985a; Correia et al., 1987; Farr et al., 1990). We and others have proposed that the Mg2+ moiety of MgGTP, but not MgGDP, interacts with specific residues in the P-subunit, and that this interaction underlies the conformation difference between tubuhn : MgGTP and tubulin: MgGDP (Sternlicht, et al., 1987; Correia et aE., 1987). If correct, the structural differ-

Tubulin binds two guanine nucleotides tightly: one exchangeably at a site in the B-subunit (E-site) and one non-exchangeably at a site presumably in the a-subunit (Geahlen & Haley, 1979; Nath et al., 1985). Nucleotide binding to the E-site is important for the regulation of microtubule assembly and function. For example, GTP in the E-site stimulates microtubule assembly whereas GDP destabilizes microtubules. Such competition results in a complex phenomenon at microtubule ends, called “dynamic instability”, believed to be essential for a variety of cellular processes including kinetochore capture, chromosome movement and division axis orientation (Mitchison & Kirschner, 1984; Walker et al., 1988; Mitchison, 1988; White & Hyman, 1987). f Author addressed.

to whom

0022~2836/92/170307-15

all correspondence

$08.00/O

should

mutagenesis;

be

307

0

1992 Academic

Press Limited

308

G. W. Parr

and H. Sternlicht -

ences between tubulin : MgGTP and tubulin : MgGDP apear to be subtle. Thus, for example, limit proteolysis and circular dichroism measurements have revealed no significant differences between the GTP and GDP bound forms although these techniques do indicate significant differences between nucleotide-bound and nucleotide-free tubulin (Maccioni & Seeds, 1983; Manser & Bayley, 1987). Nueleotide-binding proteins often utilize acidic amino acid residues to associate with nucleotides. One interaction of this type is illustrated by the preferential binding of guanine nucleotides by both EF-Tu and p21 ras (Clark et al., 1990; Pai et al., 1990). Here, an aspartic acid residue is thought to form hydrogen bonds with the endocyclic N-l nitrogen and/or the exocyclic amine at N-2 of the guanine moiety. This kind of hydrogen bonding has also been observed for a glutamic acid residue in the guanyl-specific ribonucleases, i.e. RNases Bi, Pb, and Sa (Sevecik et al., 1990). Most proteins bind nucleotide with Mg2 + co-ordinated to six oxygen atoms donated by p and y-phosphates from the nucleotide, amino acid residues from the protein and water. Typically one of the protein ligands is an acidic amino acid. In GTP-binding proteins such as EF-Tu and p21 ras an aspartic acid residue interacts with the Mg2+ moiety (Clark et aE., 1990; Pai et al., 1990). In ATP-binding proteins such as adenylate kinase, phosphofructokinase and others, both aspartic and glutamic acid residues have been observed as ligands of the Mg2+ moiety (Fry et al., 1985; Shirakihara & Evans, 1988). Thus, acidic residues appear to be a common feature of nucleotide binding. If so, then aspartic and glutamic acid residues may play similar roles in the binding of guanine nucleotides to tubulin. We undertook a site-directed mutagenesis study involving seven highly conserved aspartic acid residues (Asp67, 177, 197, 203, 209, 224 and 295) and one highly conserved glutamic acid residue (Glu69) in the putative nucleotide-binding domain of fl-tubulin (residues - 1 to 300). This domain was previously assigned on the basis of proteolysis and chemical modifications of the protein and ultraviolet crosslinking of E-site GTP (Maccioni & Seeds, 1983; Nath et al., 1985; Hesse et al., 1987; Linse & Mandelkow, 1988; Shivana & Himes, 1991; Roach & Luduena, 1984; Little & Luduena, 1987; Sternlicht et al., 1987). Mutant B-tubulins were expressed in and their nucleotide-binding properties vitro analyzed using an h.p.l.c.-based? assay (Yaffe et al., 1988a, 1989; Farr et al., 1990). We anticipated that this study would facilitate a structural under-

7 Abbreviations used: h.p.l.c., high pressure liquid chromatography; PB, a microtubule stabilizing buffer (pH 67) consisting of @l M-%(N-morpholino)ethanesulfonic acid, 2 mM-EGTA, @l mM-EDTA, 2 mM-/%mercaptoethanol, @5 mM-Mgcl,; MTP, bovine microtubule protein; MAPS, microtubule associated proteins; SDS/PAGE, sodium dodecyl sulphate/ polyacrylamide gel electrophoresis; kb, lo3 bases; bp, base-pair(s); BSA, bovine serum albumin.

standing of the E-site and elucidate the relationship between tubulin and other nucleotide-binding proteins. Our results indicate that the mutations had diverse effects on nucleotide binding. Because of its relatively specific effect on MgGTP binding, one of these mutations has identified a potential ligand for the Mg2+ moiety. This mutation at Asp224 is in a region of j%tubulin that has sequence homology with ATP-binding proteins. These findings suggest that tubulin, a GTP-binding protein, is evolutionarily related to the ATP-binding proteins.

2. Materials

and Methods

(a) Materials GTP (IIS), GDP (type I) and XTP (grade I) were purchased from Sigma. Nucleotide stock concentrations were determined spectrophotometrically. Purity was assessed by anion-exchange h.p.1.c. (GTP stocks were -4% GDP and - 1 y. GMP; XTP stocks were -3% -2 yo XMP and contained no detectable guanine XDP, nucleotides ( < @l O/o)). Chymotrypsin was purchased from Worthington Biochemicals and assayed for trypsin contamination using insulin /?-chain as described by Titani et al. (1982). Taxol, a gift from the National Cancer Institute, was prepared as a 4 mM stock solution in dimethyl sulfoxide and stored at -20°C.

(b) Preparation

of microtubule

protein

Microtubule protein (MTP) was isolated by 2 cycles of assembly/disassembly from bovine brains following temperature-based procedure (Sternlicht & Ringel, 3979) modified from Gaskin et al. (1974) and stored in liquid nitrogen in PB buffer containing 5 M-glycerol. For the heterodimer formation assay (below) the MTP was thawed on ice, diluted 1 : 1 with PB buffer and clarified at 80,000 g for 20 min at 4°C. For the taxol-dependent coassembly assay of purified heterodimer (below) the MTP was thawed on ice and used directly.

(c) DNAs

and

enzymes

Oligonucleotides for site-directed mutagenesis and priming of DNA sequencing were synthesized on an Applied Biosystems automated DNA Synthesizer in the Case Western Reserve University Molecular Biology Core Facility. Restriction and DNA modyfying enzymes were purchased from Boehringer Mannheim Biochemicals and Bethesda Research Laboratories. T4 DNA polymerase, T4 DNA ligase and T4 gene 32 protein were purchased from Bio-Rad Laboratories. Sequenase for doublestranded DNA sequencing and pTZlSR, a phagemid vector, were purchased from United States Biochemicals Corp. Standard manipulations of DNA were performed as described by Maniatis et al. (1982) unless otherwise specified.

(d) SDS/PAGE

and, fluorography

Proteins were electrophoresed on 9 or 12% (W/V) SDS/polyacrylamide gels following Laemmli (1970). The gels were fixed in 10% acetic acid, 25% methanol (v/v) for 30 min and treated with Amplify (Amersham Corp.) to enhance the resulting fluorograms.

~-T&din (e) Construction

GTP-binding

of pTZl9P

pGEM4P5 (Yaffe et aE., 1988a) was digested with @&I and Hind111 to generate the 15 kb chicken p2 tubulin cDNA containing 87 bp 5’ to the initiating ATG and 84 bp 3’ to the TGA termination codon. This 1.5 kb fragment was purified, ligated into the multiple cloning co& site of pTZ19R and transformed into Escherichia D1210. Recombinant clones were selected by restriction enzyme digestion of mini-prepared colonies. The resulting plasmid, pTZ198, contains the fi-tubulin cDNA immediately 3’ to a T7 RNA polymerase promoter and will direct the synthesis of full-length sense fl-tubulin mRNA. In addition, this plasmid contains the phage fl origin of replication and as such will replicate single-stranded when superinfected with helper phage M13K07. (f) Site-directed

mutagenesis

pTZ19j was used for the site-directed mutagenesis of fl-tubulin following Kunkel et al. (1987) and procedures outlined in the Bio-Rad Muta-Gene Phagemid Kit instruction manual. Three modifications of the Bio-Rad procedure were required. First, the superinfected phagemid in CJ236 were grown for at least 18 h to increase phagemid yields. Second, only the polyethylene glycol-precipitated phagemids on the wall of the centrifuge tube were collected to avoid a large contaminating E. coli pellet. Finally, DNA prepared from transformed E. coli MVl190 was inadequate for double-stranded DNA sequencing, and was therefore reintroduced into E. coli D1210 prior to sequence analysis. (g) In vitro

transcriptions

mRNA containing the complete coding sequence of wild-type or mutant p-tubulin was transcribed in vitro using plasmid pTZ19fl and its modified forms as previously described for pGEM4/?5 (Yaffe et al., 1988a). (h) In vitro translations p-Tubulin mRNA was translated in micrococcal nuclease-treated rabbit reticulocyte lysate (Promega), for B h at 30°C and prepared for further analysis as previously described (Yaffe el al., 1988a). Unincorporated j35S]methionine and low molecular weight contaminants were generally removed by centrifugation through 2 sequential 5-ml Sephadex G-25 spin columns as described (Yaffe et al., 1988a,b). (if Anion-exchange

h.p.Z.c.

Following Sephadex G-25 chromatography the translation reactions were applied to a Mono-Q anion-exchange h.p.1.c. column (Pharmacia LKB Biotechnology Inc.) and elnted with a linear 40 min gradient of 0 to 1 M-NaCl in 20 mM-sodium phosphate, pH 68 (Yaffe et al., 1988a,b). These conditions are sufficient to separate the 3 molecular forms of B-tubulin generated in the in vitro translation (Yaffe et al., 1988a). (j) Heterodimer

formation

assay

Translation reactions (80 ~1) were supplemented with bovine MTP to -2 mg/ml or bovine serum albumin (BSA) to -5 mg/ml. Heparin (50 pgjml final concentration) was added to inhibit microtubule assembly (Sternlicht & Ringel, 1979; Yaffe et al., 1988a,b). Samples were

309

Drain

then incubated at 37°C for 30 min and assa,yed for the relative amount of heterodimer @III) formed by anionexchange h.p.1.e. in the presence of @5 mM-MgCl, and 1.0 mM-GTP (Fig. 1). (k) TaxoZ-dependent

coassemhly

assay

Following Sephadex G-25 chromatography, translation reactions (@2 to 1% ml) were supplemented with bovine MTP (1 or 2 mg/ml final concn) and 50 pg heparin/ml, incubated at 37°C for 30 min to facilitate exchange into the heterodimeric form, and the heterodimer @III) purified on the Mono-Q column in the presence of @5 m&r-Mgcl, and 1.0 mM-GTP. After dialysis overnight against PB buffer containing 2.5 M-glycerol and @2 mM-PMSF the purified heterodimer was brought to -4 mg/ml bovine MTP and assayed by 2 cycles of taxoldependent assembly/disassembly as previously described (Yaffe et al., 1988a,b). Specific activities at each point of the assembly were calculated as 35S cts/min per mg of total protein and normalized relative to the initial sample taken as 100% (Fig. 2). In other studies (Fig. 4), translation reactions (200 pl) were brought to -4 mg/ml bovine MTP and directly assayed for assembly-competent /%tubulin by 2 cycles of taxol-dependent, assembly/ disassembly. Here specific activities were calculated from SDS/PAGE fluorograms as the integrated densities of the /?-tubulin (M, -50 kDa) bands per mg of total protein loaded. The coassembly studies (Figs 2 and 4) were typically completed within 12 h. (1) Limit

chymotrypsin

digests

Aliquots ( - 50,000 cts/min) of purified heterodimer were supplemented with BSA to -5 mg/ml and GTP to 1 mM, brought to 20 pg chymotrypsin/ml and incubated at room temperature for @2 (0), 10 or 40 min. The reactions were quenched by addition of an equal volume of SDS/PAGE sample buffer and boiling for 5 min. The samples were then analyzed by SDS/PAGE fluorography on a 12% gel (Fig. 3). In other studies translation reactions (50 ~1) were supplemented with bovine MTP to -2 mg/ml and 50 pg heparin/ml (final protein concn - 15 mg/ml) and incubated at 37°C for 30 min. Equal volume samples were then incubated with 20 /lg chymotrypsin/ml and processed for fluorography as described above. (m) Nucleotide

binding

assay

The apparent K,, values of P-tubulin for various nucleotides were determined as previously described using an anion-exchange h.p.l.c.-based assay (Farr et al., 1990). Measurements of the E69Q and D203N heterodimers were accomplished by first incubating the initial translation reactions (500 pl) at 37°C for 30 min with an equal volume for -4 mg/ml bovine MTP containing - 100 pg heparin/ml. This incubation greatly increased the relative amount of pII1 and facilitated the analysis of the heterodimer. Following incubation, the samples were passed through 6 sequential 5-ml Sephadex G-25 spin columns and then assayed. In all other cases the initial translation reactions were passed through 6 Sephadex G-25 spin columns and assayed directly (Farr et al., 1990). Binding studies in the presence of Mg2+ were done with @5 miw-MgCl, in the running buffers except in cases where our preliminary measurements indicated that the Kd values exceeded 0.2 IIIM. In these cases MgC1, concentration was increased to 1.0 to 5.0 mM as needed to ensure sufficient Mg2 + during the assay to complex nucleotide.

G. W. Farr

310

and H. Sternlicht

Table 1 Mutations

D67N E69Q D117N D197N D203N D209N D224N D295N

introduced

into B-tubulin$ domain

GTP-binding

GTP crosslinked to region 63-77 in B-tubulin (Linse & Mandelkow, 1988) GTP crosslinked to region 63-77 in B-tubulin (Linse & Mandelkow, 1988) No known homology No known homology Putative Mg ‘+ binding site (region homologous to GTP-binding proteins; Sternlicht et al., 1987) No known homology Putative Mg ‘+ binding site (region homologous to ATP-binding proteins) Putative guanine binding site (region shows reverse homology to GTP-binding proteins; Sternlicht et al., 1987)

200

D209N

R

Single letter abbreviations are used to represent amino acid residues. Numbers give the residue position in the primary structure of fi-tubulin. The first letter indicates the original residue; the last letter indicates the mutation. See Discussion for further details.

Binding studies in the absence of Mg’+ were done with 1.0 mM-EDTA in the running buffers to complex residual Mg*+ (Higashijima et al., 1987). Nucleotide binding affinities of wild-type were measured in triplicate (Parr et aZ., 1990) while those of the mutants were typically done in duplicate although some were single determinations (Tables 3 and 4).

synthesis

80

20-O

of mutants

Table 1 lists eight mutants constructed for this study. Five of these mutations were selected on the basis of putative homologies with other nucleotidebinding proteins and three were in regions of no 35S-labeled B-tubulin mutants known homology. and wild-type controls were synthesized in rabbit reticulocyte lysates as previously described (Yaffe et al., 1988a; Materials and Methods). SDS/PAGE confirmed that mutant and wild-type translation reactions gave similar amounts of radiolabeled protein ( >80°h of total protein was full-length fi-tubulin, data not shown).

25.0

D224N A

40

3. Results (a) In vitro

15.0

0 80

D197N

4c C 2c IC

31

C 3c 2c IC

(b) Heterodimer

formation

We previously reported that assembly-competent tubulin is produced when wild-type B-tubulin is translated in reticulocyte lysates. The protein was found in three distinct molecular forms separable by either anion-exchange (Mono-Q) or size-exclusion chromatography: a high molecular weight complex of /?-tubulin and a lysate protein(s) (PI), the free b-subunit @II), and the heterodimeric complex of /?-tubulin with residual a-tubulin from the lysate @II). Incubation of the translation reactions with bovine microtubule protein gave an increase in the relative amount of heterodimer. This increase was the result of an exchange reaction where PII replaced the P-subunit in bovine heterodimer

I

C 20.0

25.0

Time (mini

Figure 1. Anion-exchange chromatography of the b-tubulin mutants. 80 ~1 portions of the translation reactions were incubated with an equal volume of either BSA (10 mg/ml)( --De ) or MTP ( -4 mg/ml) (a=) for 30 min at 37°C and centrifuged through 2 Sephadex G25 spin columns. The effluents, which contained essentially the same amounts of total fuli-length P-tubulin, were applied to a Mono-Q column and eluted in 20 mM-sodium phosphate (pH 6.8) using a 0 to 1 M-Nacl gradient in the presence of @5 mM-MgCl, and 1.0 mM-GTP. Numerals I, II and III denote the elution positions of the PI> j3II and 0.55 and 0.62 M-NaC1, PI11 polypeptide forms at -0.46, respectively, based on wild-type P-tubulin (Yaffe et al., 1988a) (see the text).

p-Tub&n

GTP-binding

311

Domain

Table 2 Recovery of /?-tub&n

forms from the Mono-Q column

Cts/min recovered (minus background)

Mutant

BID

Total

291,000 41,700

78,900 560,000

443,000 618,000

49,400 11,000

0 16,900

49,400 27,900

7900 5000

347,000 58,300

4500 36,700

359,000 100,000

47,300 17,800

145,000 34,200

78,800 285,000

265,000 337,000

76,600 11,900

36,400 3400

78,200 221,000

191,000 236,000

21,600 8200

27,800 5000

0 28,800

49,400 42,000

43,100 18.700

279,000 82,400

55.800 283,000

378,000 384,000

21,000

148,000 32,500

12,800 201,000

182,000 234,000

370,000 174,000

17,900 174,000

409,000 358,000

PI

P

wt

+ BSA + MTP D67Nt +BSA +MTP E6QQ + USA + MTP D177N + mL4 + MTP D197N + USA f MTP 9203N + BSA + MTI’ D209N + BSA + MTP D224N + USA + ,MTP D295N + USA + MTP

73,300 16,300

0 21,100 10,100

Derived from Figure 1. The translation reactions applied to the Mono-Q column contained the same amount of full-length /%tubulin (_+ 10%) as determined by SDS-PAGE fluorography. wt, wild-type. t Translation reactions shown in bold were also analyzed by coassembly with MTP (see text below). $ Not detectable due to large background.

producing the hybrid heterodimer a(bovine)fl(chick) (Yaffe et al., 1988a, 1989; also Fig. 1, top panel). We investigated whether the mutant fi-tubulin polypeptides produced in reticulocyte lysates could also form heterodimers. The translation reactions of wild-type and mutants were incubated with BSA or MTP and then chromatographed on a Mono-Q anion-exchange h.p.1.c. column. Samples were eluted using a salt gradient (0 M to 1 ix-Nacl) supplemented with high concentrations of GTP and MgCl, to optimize recovery (Yaffe et al., 1989; Farr et al., 1990; Materials and Methods). The results are presented in Figure 1 (ordered according to the relative amount of fi-tubulin recovered) and Table 2. When the mutant translation reactions were incubated with BSA, the free /&subunit, BII, was generally the major product (Fig. 1, open squares). In contrast, when the mutants were incubated with MTP, the heterodimer, BIII, was generally the major product (Fig. 1, closed squares). results. When Dl97N and E69Q gave unusual D197N was incubated with BSA, the major products were /31 and /?TII rather than PII. When E69Q w-as incubated with MTP, the major product was BII. rather than BIII, although the intensity of

O----j

s2 Cycle

Figure 2. Coassembly of the purified mutant heterodimers. The heterodimers (~111) of wild-type and mutants were isolated from translation reactions by anionexchange chromatography then supplemented with MTP (-4 mg/ml) and carried through 2 cycles of taxol-based assembly/disassembly (Materials and Methods). Portions containing equal amounts of total protein from consecutive cycles of assembly/disassembly were analyzed by SDS/PAGE fluorography. The data are shown as a percent of the specific activity recovered at the the initial sample and are plotted as a function of cycle position for wild-type (a-), E69Q (-++-), D177N (-o-o-), D197 (+-+), D203N (+a-), D209 (u), D224N (a-+) and D295K (+&-). I.S., Pl, Sl, P2 and S2 denote, respectively, the initial sample, the first pellet, the first supernatant (end of cycle 1); the second pellet, the second supernatant (end of cycle 2).

the /XII peak was reduced significantly. Nevertheless, both of these mutants did form beterodimer upon incubation with MTP (Fig. 1). These findings demonstrated that mutant PIIs, like wildtype PII, were capable of associating with a-tubulin to form heterodimers. (c) Coassemblylproteolysis of pur$ied mutant heterodimers We investigated whether the mutant heterodimers recovered from the column were capable of coassembly with carrier MTP (Fig. 2). A study of D67X was not possible because of the poor recovery of this mutant’s heterodimer. Mutant and wild-type heterodimers isolated on the Mono-Q column were supplemented with MTP and carried through two cycles of taxol/Ca’+ assembly/disassembly (Fig. 2; Materials and Methods). Previous studies with wildtype tl and P-tubulin established that this stringent coassembly procedure can distinguish between specific incorporation of radiolabeled subunits into the microtubule lattice and non-specific interactions with the microtubule (Yaffe et al., 1988a,b). As a result, this assay quantitatively distinguishes between functional and non-functional protein. Wild-type heterodimers showed an approximately 1.5fold increase in specific activity between

312

(2. W. Farr

and H. SternEieht D203N

Wt

(E69Q, Dl77N, D197N, D209N, D224N, D295N 1 0

IO min

40 min

IO min

40 min

Figure 3. Limit proteolytic cleavage of the purified mutant heterodimers. Portions of purified wild-type and mutant heterodimers (-50,000 cts/min) isolated from the Mono-Q column were digested at room temperature for 10 s (“O”), 10 min or 40 min in the Dresence of 20 ue chvmotrvnsin/ml (Materials and Methods). Digests were analyzed by SDS/PAGE fluorography. A .”

”

“I

the initial sample (1,s.) and the supernatants from the first (Sl) and second (52) assembly/disassembly cycles (Fig. 2). This increase reflected the loss of the MAP component of the MTP and is indicative of 100% assembly-competent tubulin (Yaffe et al., 1988a). With the exception of D203N, the mutants were also assembly-competent. Compared to the wild-type control - 90% of D295N, - 85% of D209N, -7Oo/o of E69Q, D177N and D224N, and -60% of D197N survive as competent protein through the two cycles of coassembly. In contrast, <20% of the D203N heterodimers were assemblycompetent (Fig. 2). The purified mutant and wild-type heterodimers were further investigated by limit proteolysis with chymotrypsin (Fig. 3). Mutants E69Q, D177N, D197N, D209N, D224N and D295N gave digestion patterns indistinguishable from wild-type. These and 34 kDa fragments digests showed the -22 characteristic of native tubulin (Yaffe et al., 1988a; Brown & Erickson, 1983). In contrast, D203N heterodimer was highly sensitive to chymotrypsin, a result consistent with its low assembly competence (Fig. 2). D67N heterodimer was not examined because of its poor recovery. We concluded that the mutant heterodimers recovered from the column, with the exception of D203N and possibly D67N, contained primarily assembly-competent protein with conformations similar to wild-type. (d) Coassemblylproteolysis translation

of the unpurijied products

We observed that the mutants were recovered from the Mono-Q column at levels often signifi-

cantly lower than wild-type (Fig. 1 and Table 2). Mutations D67N and D203N, for example, were recovered poorly, D117N, D197N and D224N gave intermediate levels of recovery and D209N and D295N gave recoveries that were similar to wildtype (compare the BSA containing samples in Fig. I and Table 2). To elucidate the basis of these differences we examined the ability of wild-type and several mutants (D67N, D203N, D224N and D295N) to coassemble with MTP directly from the translation reactions (Fig. 4). In contrast with the purified heterodimer, the coassembly of wild-type B-tubulin from the translation reactions showed an approximately twofold reduction in specific activity between I.S. and Sl or S2. We interpret this observation as evidence that wild-type translation reactions generate two populations of newly translated polypeptide chains: an “assembly-incompetent” and an “assembly-competent” fraction each representing -l/2 of the total fi-tubulin synthesized. The mutants behaved in a similar manner (Fig. 4) suggesting that these translation reactions also contained two populations of fi-tubulin poiypeptides. However, the initial drop in specific activity between I.S. and Sl was much greater - 75 to 95% of the mutant indicating that P-tubulin polypeptides were assembly-incompetent. If the b-tubulin translation reactions are ordered according to increasing assembly-competence, then this order D67N; D203N, D224N; D295N; and wildtype follows a similar trend as the order of recovery of their heterodimers from the Mono-Q column (Fig. 1 and Table 2, recoveries of /?I11 (+MTP)). The above result, taken together with the results for the purified heterodimers, would imply that the

fl-Tubulin

GTP-binding

313

Domain

therefore be composed of conformationally altered which bound (denatured/misfolded?) proteins tightly to the column. Limited proteolysis studies supported this interpretation (data not shown). Translation reactions of wild-type, D67N, D203N, D224N and D295N were supplemented with MTP carrier protein and then digested with chymotrypsin for varying periods of time (Materials and Methods). Greater than 50 y. of wild-type but < 5 to 20% of the mutant /3-tubulin polypeptides in the translation reactions remained full-length after a digestion with ehymotrypsin$. 40 minute Furthermore, D67N and D203N, which showed the lowest recovery on the column (Fig. 1); also showed the greatest sensitivity to chymotrypsin. Cycle

Figure 4. Coassembly of the unpurified p-tubulin mutants. s%&labeled translation reactions (wild-type and mutants) were supplemented with MTP protein carrier ( -4 mg/ml) and carried through 2 cycles of taxol-based assembly/disassembly (Materials and Methods). Portions containing equal amounts of total protein from consecu-

tive cycles of assembly/disassembly were analyzed by SDS/PAGE fluorography. The data are shown as a percent of the specific activity recovered at the initial ), D203N sample for wild-type (-<>-o- ), D67N ( ++x-

(--@+-J--), D224N (++--)

and D295N (e)

and

are plotted as a function of cycle position. Cycle positions denoted as in Fig. 2.

assembly-competent fractions from the translation reactions (wild-type, D67N, D224N, D295N and possibly D203N) were preferentially recovered from the column whereas the assembly-incompetent fractions were mainly lost to the column. If correct, this interpretation would largely account for the differences in recoveries between these mutants and wildtype (Fig. l)?. D203N behaved anomalously. When D203N and D224N were assayed directly from the unpurified translation reactions they showed similar levels of assembly competence (Fig. 4). In contrast, when these mutants were assayed following purification on the Mono-Q column N 70 y. of the D224N and 120% of the D203N heterodimers were assembly-competent (Fig. 2). The lower percentage of assembly-competent heterodimer for D203N may reflect a copurification of assembly-incompetent protein on the Mono-Q column and/or a reduced stability of the D203N heterodimer. Previous studies have shown that the recovery of tubulin from the Mono-Q and other h.p.1.c. columns depend on the presence of nucleotide in the E-site and appeared to be affected by conformation-dependent hydrophobic interactons between the protein and the column (Farr et al., 1990; Hanssens et aZ., 1990). The assembly-incompetent fractions might t A detailed understanding of the mutant recoveries would require a study of the intrinsic recoveries of each of the mutant j%tubulin forms. These studies were not done.

(e) Nucleotide

binding

The nucleotide binding affinities of tbe mutants were estimated from analyses of PII and ,811I recoveries on the Mono-Q column (Farr et aE., 1990; Materials and Methods). This assay exploits the properties of newly translated p-tubulin and requires (1) that P-tubulin polypeptides (mutant and wild-type) undergo differential losses on a Mono-Q anion-exchange column, which depend on nucleotide binding at the E-site (loss of the nucleotide-free polypeptides > loss of nucleotidecontaining polypeptides); (2) that nucleotides in the running buffers be in dynamic equilibrium with the E-site during protein elution. The method is highly sensitive and gives apparent Kd values for nucleotide binding to wild-type tubulin that are in good agreement with published values (Farr et al., 1990; see Discussion). 35S-labeled wild-type and mutant b-tubulin were synthesized in reticulocyte lysates and treated as described (Materials and Methods). Portions were loaded on the Mono-Q column with various fixed concentrations of nucleotide in the running buffers permitting simultaneous measurement of the binding constants for the free monomer and the heterodimer (mutants D117N, D197N, D209N, D224N and D295N). This approach is illustrated in Figure 5 for GTP binding to wild-type and D224N fi-tubulin in the presence and absence of magnesium. In some cases the translation reactions were first incubated with MTP to exchange PII into /?I11 facilitating measurement of the heterodimer (mutants E69Q, D67N and D203N). Table 3 lists the apparent Kd values of wild-type and mutant heterodimer for MgGTP and GTP; MgGDP and GDP; and MgXTP (the latter served as a measure of guanine binding specificity (Farr et al.., 1990)). Table 4 lists the corresponding apparent Kd values of the free monomer. Mutant j%tubulin monomers and heterodimers bound nucleotides with similar specificity demonstrating as was shown for wild-$ This study was done with a lower chymotrypsin total protein ratio than the purified heterod.imers (Fig. 3) (Materials and Methods) to faeilitate the analyses.

to

314

6. W. Farr and H. Sternlicht 60

20

0 n

.E E \ e u U-J 2 40-

0

I/[GTP]

t/d’)

5.0

(cl A

15

20 Time

25

0 I/[GTPI

(min)

(mu-‘)

30 m t‘ 0”

20

0 lo0 ” .c E > t

IO G4 x .E E \ 2 0 m 2

0

4 -

12

6

0 I /[GTP]

: P 0 x

40

( mt.+-’ )

20

I/[GTP]

Time (min)

(rnM-’

)

Figure 5. Nucleotide binding assay based on enhanced recoveries from the Mono-Q column. Elution profiles of wildtype tubulin ((a), (c)) and mutant D224N ((e), (g)) in the presence of 0.5 mM ((a), (e)) or absence of ((c), (g)) MgCl, are shown for several fixed concentrations of GTP in the column running buffers. Double reciprocal plot analysis of the elution profiles are shown for wild-type with and without MgCl, in (b) and (d), respectively, and mutant D224N with and without MgCl, in (f) and (h), respectively. 1 mM-EDTA was added to the running buffers to complex residual Mg2’ increase in the recovery of radiolabel at the PII and /?I11 impurities in (c) and (g). (a), (c), (e ) and (g) show the progressive positions with increasing GTP concentrations for a single experiment (to facilitate presentation only 3 of the GTP concentrations used are shown). The concentrations of GTP in the running buffers were as follows: (a) a-, 0 mu; --@+, 25nM; w, 200nM; -)-t, 5OOniu. (c) w, OHM; e, 5 PM; -DI)-, 10~~; -cf, 20 PM. U-U-,

(e)

-o-@

10

PM;

> 0 PM;

s,

-+.-8.5

100

PM; PM.

Double

m-,

reciprocal

1 PM;

plots

-#-+->

of this

10 PM.

increase

(8)

s,

provide

oPf/r;

estimates

-.--.-,

of the

5 WM[;

apparent

P-Tub&n

GTP-binding

315

Domain

Table 4

Table 3

Apparent dissoeia~~m~

Apparent dissociation constants (K,,) for tubulin

wtt

+Mg’+ -Mg’+ D67N + Mg’+ E69Q + Mg’+ -Mg2+ D177N +Mg’+ -Mg’+ Dl97N + Mg’+ -Mg2+ D203N +Mg2+ -Mg’+ D209N +Mg’+ -Mg2+ D224N + Mg’+ -Mg’+ Exchanged +MgZ+ -Mg2+ D295N +Mg’+ -Mg’+

GDP

XTP

40f4 fJ1*10

1700+500

GTP

XTP

GDP

wtt lo+1 8100~600 >lx106 40+ 12: 390+715

> 1 x 106 5Ok 161 96 f 141

>lx106 3100+ 7001

lSO&35:: 480 5 O$ 15+6$ 400 & 163 a900+200$ 2.2 x lo6

29,000 19,000

>5x

106

2o*q 1400+ 3001 930 k 251 2600 + 4501 D224NI/ 1500f 1001 4600+3300$ 24+8$ 2000+450$

660 & 251 820+ 480$

32f2$ 32+16$

for free

& @M)

K, (W GTP

(I&)

-su uni s

heterodimer

+Mg’+ -Mg’+ E69Q +Mg*+ -Mg2+ D177N +Mg2+ +Mg’+ D197N +Mg2+ -Mg’+ D209N +Mg’+ -Mg2+ D224N +Mg2+ -Mg’+ D295N +Mg’+ -Mg2+

39+5 1600+400 97 * 19$ 98Ok 1605

68* 10 140+30

12,000+2000

50,000

75+1$ 280&90$

80 1000+780$ 69*20$ 140&80$ 38f4$ 3600 F 20003 1500+350$ 3600+800$

860 f 252 850 k 501

-4.9 x lo6

32 k 14$ 15OOf510$

46+3 32_+ 141

39,000~27,090$

-3x106 Determined Methods. t Wild-type jn=2. gn=3.

as described done in triplicate

in Figure

5 and Materials

and

(Farr et al.. 1990).

4500* 1500$

Determined as described in Figure 5 and Materials and Methods. t Wild-type done in triplicate (Farr et al., 1990). gn=2. §n=3. 11These apparent K, values were measured using D224N translation reactions preincubated with bovine MTP to introduce an excess of assembly-competent protein (see the text).

type, that association with the a-subunit does not cause major effects on nucleotide binding (Farr et al., 1990). The mutations gave six different effects on nucleotide binding (Table 5). Furthermore, both the heterodimer and the free monomer for each mutant showed the same effects. D67N abolished any detectable nucleotide binding (see Discussion). E69& and D197N specifically enhanced GTP binding in the absence of Mg*+ 20-fold. D177N also enhanced GTP binding in the absence of Mg2+ -26fold but weakened GTP binding in the presence of Mg ‘+ l&fold. D209N and D295N bound guanine nucleotides with affinities that were similar to wild-type. D203N caused a general shift in Kd values. Affinities for the nucleotides, for example, were reduced - 200 to 700-fold relative to wild-type

fl-tubulin (Table 3). Nevertheless, the distinctive features of the E-site, e.g. its requirement for Mg2+ in GTP binding, the preference of MgGTP over MgGDP and the high binding specificity for guanine nucleotides, were retained. D224N reduced p-tubulin’s affinity for MgGTP - loo-fold relative to wildtype while having little effect on GTP binding in the absence of Mg ‘+ (Tables 3 and 4; Fig. 5 E to H)?. The net result was almost to abolish the Mg2+ requirement for GTP binding. In contrast, MgGDP and GDP binding were perturbed to similar degrees by the mutation (Kd values increased 10 to 15-fold relative to wild-type). Measurements for GTP binding to D224N in the presence and the absence of Mg2+ were repeated using preparations that were enriched - IO-fold for assembly-competent heterodimers. This was accomplished by preincubating the translation reaction (500 pl, -30 nna total p-polypeptides synthesized) with MTP (250 ~1, 3.5 mg/ml bovine heterodimer) at 37°C for 36 minutes before measuring nucleotide binding. These enriched preparations gave apparent Kd values in good agreet This perturbation Mg 2+ is also observed specificity is retained

of GTP binding in the presence of for XTP indicating that guanine (Tables 3 and 4).

dissociation constant (Rd) for GTP binding to /?I1 ( m) and /III1 ( -+c ) w h ere l/A cts/min = (&/A cts/min,,,) (I/[GTPl) + I/A ctsimin,,,), or apparent Kd = slope/y-intercept (Farr et al., 1990). A cts/min denotes the increase relative to the “0” GTP control; A cts/min,,,, the corresponding increase in radiolabel at “infinite” concentrations of GTP. These plots show all of the GTP concentrations used for each experiment. The data were analyzed by linear regression. (f) and (h), a---, I/A cts/min= x 10’; ++--; l/A cts/min= x 104).

316

G. W. Farr and H. Sternlicht

Table 5 EJIlfectof mutations on nudeotide binding to /?-tubulin Mutants

Effect on nucleotide

binding

D67N E69&, D197N

Abolishes nucleotide binding Enhances affinity for GTP in the absence of Mg*+ (GTP binding in the presence of Mg’+

D177N

Enhances affinity for GTP in the absence of Mg’+, diminishes affinity for GTP in the presence of Mg*+ Nucleotide binding similar to wild-type Diminishes affinity for all nucleotides (no effect on specificity) Diminishes affinity for GTP in the presence of Mg’+ (no effect on GTP binding in the absence of Mg’ + )

similar to wild-type)

D209N, D295N D203N D224N

ment with those obtained directly translation reactions (Table 3).

from

tiyd-X-Arom-X-Acidic Mg2+ Brtding loop

ATPase/Klnase sovinaATPose

”

696

the

SH2 *

LLFID

N

IC ROG-FPNR

WFOE

720 F

IC

K

ILYQD

,A

RW

SHI *

R C NGVLEGIR c KC

LKYNE

200 F C

4. Discussion We have carried out a site-directed mutagenesis study of the putative GTP-binding domain of characterB-tubulin. Biochemical/biophysical izations of mutant proteins typically depend on in vivo expression systems to produce high levels of protein. Unfortunately, attempts to overexpress tubulin have led either to insoluble/denatured protein, as in E. coli (Yaffe et al.: 1986; Wu & Yarbrough, 1987), or to lethality, as in yeast (Burke et al., 1989). In an alternative approach we used in vitro translation in rabbit reticulocyte lysates to generate small amounts of highly radiolabeled mutant b-subunits (Table l), which could be assayed sensitively for their ability to form tubulin heterodimers (Fig. l), to coassemble with MTP into microtubule polymer (Figs 2 and 4), and to bind nucleotides (Fig. 5; Tables 3 and 4). This approach is supported by a body of literature demonstrating the utility of reticulocyte lysate systems for studying mutant proteins (cf. Abbott & Feizi, 1991; Dalman et al., 1991; Vorburger et al., 1989), as well as our studies of wild-type CIand /I-tubulin synthesized in reticulocyte lysates (Yaffe et al., 1988a,b, 1989; Farr et al., 1990). (a)

nucleotide-binding

proteins

and

three

were

from regions with no known homololgy. fi-Tubulin has three regions with apparent homology to the nucleotide-binding

GTP-binding We

MVL

IC ORTLKLNP PSYGD TT PTFGD IC F

YC

IC

YC

IC

Figure 6. Sequence homologies

F

with

225 L

TT PTYGD TT PTYGD

other

nucleotide-

binding proteins. Schematic diagrams for p-tubnlin show open bars with blackened regions representing the putadomain; hatched bars denote the tive GTP-binding C-terminal domain. (a) It has been suggested that P-tubulin contains the 3 consensus sequences for binding phosphate, Mg2+ and guanine base identified by Dever et al. (1981) for GTP-binding proteins (see tbe text). However, 2 of the sequences (*) are in the reverse (C to N-terminal) orientation (Sternlicht et al., 1987). An additional region encompassing residues 63 to ‘70: which photoaffinity labels with GTP, is also shown and is thought to be a guanine-binding site (Linse & Mandelkow, 1988). (b) fl-Tubulin contains consensus sequences for binding Mg’+ and phosphate identified in ATP-binding proteins. The potential phosphate-binding site &own in (b) contains the motif GXXGXG commonly observed in these proteins (Sternberg & Taylor, 1984; Wierenaga et aE., 1986). The potential Mg2+-binding site contains the motif “Hydrophobic-Variable-Aromatic-Variable-Acidic’~ found in ATPases, serinelthreonine kinases and myosin (Hanks et al., 1988; Burke et al., 1990). This motif bas been implicated in the binding of the Mg2+ moiety of MgATP in myosin (Burke et al., 1990), where it is adjacent to 2 critical sulfhydryl residues (SHI and SH2) and part of an extended sequence that bears homology to P-tubulin (Ponstingl et al., 1981a; Krauhs et al., 1981).

Selection of mutants for investigation

We focused on seven highly conserved aspartic acid residues and one glutamic acid residue within P-tubulin’s putative GTP binding domain (Table 1). These residues represent a subset of the 25 highly conserved acidic residues in this domain (cf. Barahona et al., 1988). Five of these residues were from regions that share apparent homology with other

,mYD

previously

consensus

sequences

of

the

proteins (Dever et al., 1987; Fig. 6(a)). speculated

that

two

of

these

sequences, zo3DNEA206 and 298NKAD2”5, are homologs for the binding sites of the Mg2+ and guanine moieties of MgGTP, respectively (Sternlicht et al., 1987). One caveat to this assignment is that sequence NKAD is presented in the reverse C to N-terminal orientation. It is difficult to see how a reverse orientation would generate the appropriate surface features permitting the residues to function as predicted. Nevertheless, consensus sequences for other types of binding interactions have been shown to function in either orientation (Chiang & Dice, 1988; Becker

& Roth,

1992). Another

sequence

of

/STubdin

GTP-binding

interest, region 60 to 70 of /I-tubulin, was noted by Leberman & Egner (1984) to be similar to a region preceding the guanine binding site of EF-TU interest (Fig. 6(a)). This region is of additional because photolabeling studies of tubulin with GTP identified a crosslink to a tryptic peptide at residues 63 to 77 (Linse & Mandelkow, 1988)t. Within this region sequence ’ 3‘NKCDl 38 in EF-Tu would correspond to sequence 67DLEP70 in /I-tubulin. Although these two sequences are very different, both could hydrogen bond to guanine via their acidic residues. #I-Tubulin also bears homology with ATP-binding proteins. Ponstingl et aZ. (1981a,b) and Krauhs et al. (1981) noted significant sequence homology between a and a-tubulin and a variety of muscle proteins including actin, myosin and troponin T. In particular, the myosin head region 695 to 720, which contains two critical sulfhydryl residues SHl and SH2 important for myosin function, showed sequence homology with regions -200 to 225 in a and B-tubulin (Ponstingl et al., 1981a; Fig. 6(b)). Luduena and colleagues showed that removal of GTP from tubulin permits the crosslinking of one of the two cysteine residues in region 200 to 225 to CyslZ by the bifunctional sulfhydryl reagent N’,N’-ethylenebis(iodoacetamide) (Roach 83 Luduena, 1984; Little & Luduena, 1987). In addition maytansine, which is known to block the GTP-binding site (Huang et al., 1985b), blocked this crosslinking reaction (Roach & Luduena, 1984). These studies are consistent with a functional homology between regions 695 to 720 in myosin and 200 to 225 in P-tubulin. Furthermore, Burke et al. (1990) proposed that the myosin head region 695 to 720 is a major component of the MgATP binding site, providing a likely site of interaction for the Mg2+ moiety with acidic residues in this region. In B-tubulin, region 206 to 225 contains three highly conserved aspartic acid residues: Asp203 (discussed above), Asp209 and Asp224. The latter residue is part of a highly conserved sequence in B-tubulin to consensus (Fig. 6(b)), which is homologous sequence “Hydrophobic-Variable-AromaticVariable-Acidic” proposed by Burke et al. (1990) for ATP-binding proteins. (b) The mutation paradigm Mutations in P-tubulin could either directly perturb nucleotide binding (by direct replacement of a binding residue) or indirectly perturb binding (by an alteration in protein conformation). In view of the sparse knowledge of tubulin structure it may not be possible to unequivocally distinguish direct from indirect perturbation. However, in certain cases, we believe convincing evidence that a particular mutation has a direct effect on nucleotide binding can be obtained. Mutational studies of the i Photolabeling studies with GTP have also identified two other regions in j?-tubulin, i.e. residues 10 to 20 (Shivana & Himes, 1991) and 155 to 174 (Hesse et al., 198T)I as potential binding sites for GTP.

Domain

317

% 2+ binding

site app eared attractive since the Mg2+ moiety is essential for binding GTP but not GDP (K,(GTP) = 800 &(MgGTP); &(GDP) = 2 &(MgGTP)) (Huang et al., 1985a; Correia et al., 1987; Farr et aE., 1990; Table 3). Thus, if a mutation specifically reduced the afinity for GTP in the presence of Mg’+, without significantly affecting GDP binding, it would seem reasonable to conclude that the mutation had a direct effect on nucleotide binding. Similarly, if a mutation changed nucleotide specificity, e.g. from guanine to xanthine nucleotides, without diminishing other binding properties it would also be reasonable to conclude that the mutation had a direct effect on nucleotide binding. For example, Hwang & Miller (1987) changed specificity in EF-Tu from guanine to xanthine nucleotides by replacing the aspartic acid residue in consensus sequence NKXD with an asparagine residue. Guided by the above paradigms our results suggest that mutation D224N caused a direct perturbation of nucleotide binding at the Mg2+ site (see below). (c) Assembly-competent versus assembly-incompetent protein Coassembly and limited proteolysis studies revealed that the translation reactions generated two populations of mutant polypeptides. The major fraction, comprising 75 to 90% of the polypeptides, was conformationally altered. This conclusion is based on the fact that this fraction showed enhanced susceptibility to chymotrypsin relative to wild-type (data not shown), did not coassemble with MTP (Fig. 4) and, with the possible exception of D203N, appeared to be largely lost on the Mono-Q column. The remaining fraction of a-tubulin polypeptides could be recovered from the Mono-Q column, often at levels similar to wild-type protein. Limit chymotrypsin digestions of the recovered heterodimers suggested no or at most subtle differences in structure relative to wild-type controls (Fig. 3). Furthermore, these heterodimers were generally assembly-competent by the stringent taxol-based assay, although differences relative to wild-type were noted (Fig. 2). Excluding D203N, the mutant coassemblies at the end of 52 were 60 to 95% that of wild-type. The bases for these differences are not understood. Reduced assembly could, for example, be due to protein instability or may be an inherent property of these mutants. To resolve this uncertainty, one would have to isolate the mutants in sufficient quantity for assembly studies without carrier MTP. This is currently not feasible. The finding that the assembly-incompetent protein predominated in the translation react’ions was not unexpected as it was previously observed that wild-type /I-tubulin also contained significant amounts of non-functional p-tubulin ( - 40 to 50 y. of total synthesis; Yaffe et al., 1988a; Cleveland et al., 1978; also Fig. 4). The higher fraction of assembly-incompetent protein in the mutant translation reactions is not completely understood. This

318

G. W. Farr

and H. Sternlicht

may simply represent an accumulation of denatured or partially denatured protein as a result of decreased protein stability. However, aside from D67N and possibly D203N we have no evidence that the stability of the mutants differed significantly from that of wild-type. The origin of the assembly-incompetent proteins is likely to be complex. We have recently identified a molecular chaperone in reticulocyte lysates that is intimately associated with the biogenesis of CI and p-tubulin polypeptides (Yaffe et al., 1992). Thus, it is possible that the mutations alter to varying degrees the normal processing/folding of /I-tubulin on this chaperone. Nucleotide-binding affinities were determined using mutant translation reactions even though these preparations contained 75 to 95% assemblyincompetent protein. We cannot completely exclude the possibility that the assembly-incompetent fraction contributed to the measurements even though this fraction was preferentially lost on the Mono-Q column. With the possible exception of D203N, the assembly-incompetent proteins did not appear to interfere with the measurement of nucleotidebinding affinities. This is supported by the fact that wild-type translation reactions, which contained - 50 y0 assembly-incompetent protein (Fig. 4), gave nucleotide-binding constants that were identical to published values for tubulin dimer (Farr et al., 1990; Zeeberg & Caplow, 1979; Correia et al., 1987; Fishback & Yarborough, 1984). In addition, when an excess of assembly-competent tubulin, in the form of MTP carrier protein, was added to the translation reaction of D224N, the measured nucleotide-binding constants were unaltered (Table 3; Results). This suggests that the measured binding constants were independent of the amounts of competent or incompetent background protein eluting during chromatography. In addition, we cannot exclude the possibility that the assemblycompetent fractions were somewhat unstable (see above). Such an instability could have affected the Kd determinations. However, in contrast to the coassembly studies, which required several one hour incubations at 37 “C, the Kd determinations required only 25 minutes at room temperature for each nucleotide concentration. (d) Effects of the mutations

on nucleotide

binding

The eight acidic residues mutated in this study fell into six distinct groups (Table 5). (i) D67N Mutation D67N abolished any detectable nucleotide binding. Such a perturbation would produce a nucleotide-free P-polypeptide and could be responsible for the high levels of assembly-incompetent protein and poor recoveries found for D67N (Figs 1 and 4). This loss of nucleotide-binding is consistent with region 63 to 77 containing a binding site for the (Linaw & Mandelkow, 1988). guanine moiety because nucleotide binding was so However,

profoundly altered, an alternative possibility is that the primary effect of this mutation was to perturb protein conformation. (ii) E69Q and D197N The affinities of E69Q and D197N for GTP in the absence of Mg2+ were enhanced -20-fold without affecting MgGTP binding (Tables 3 and 4). These mutants produced heterodimers with similar levels of assembly competence (Fig. 2) and with conformations indistinguishable from wild-type based on limit proteolysis (Fig. 3). However, the recovery of the E69Q heterodimer was anomalous, suggesting a subtle but important change in its properties. The enhancement of GTP binding in the absence of Mg’+ appeared to be relatively specific even though the two mutations are greatly separated in the primary structure of j-tubulin. We do not understand the molecular basis for this enhancement and suspect that this class of mutant produces its effect via subtle and indirect perturbations of the nucleotide-binding site. In comparison with D67N, E69Q caused a relatively minor perturbation in nueleotide binding. As a result, the putative guanine-binding site in region 63 to 77 discussed above would not be expected to include Glu69. (iii) Dl77N The affinity of D177N for GTP in the absence of Mg2+ was enhanced -20-fold. In contrast to E69Q and D197N, this mutation also reduced the affinity for GTP in the presence of Mg2+ -18-fold (Table 3). The molecular basis for these effects remain to be determined. (iv) D295N and D209N This class gave nucleotide-binding constants that were similar to wild-type (Tables 3 and 4). Purified heterodimers from both mutants coassembled to a level > 85% of wild-type and gave chymotrypsin digests that were indistinguishable from wild-type (Figs 2 and 3). These observations argue that Asp295 does not participate in nucleotide binding to P-tubulin as we suggested earlier. (v) D203N

This mutation significantly diminished the ability of B-tubulin to bind nucleotide without altering the protein’s specificity for guanine nucleotides or its requirement for Mg2+ when binding GTP (Table 3). This behavior is consistent with a specific effect on the binding of the a and B-phosphate and/or ribose moieties of the nucleotide. However, the D203N study used mutant proteins that eoassembled poorly and were highly sensitive to chymotrypsin digests (Figs 2 and 3). Thus, it is possible that the observed changes in nucleotide binding for D203N are the result of a perturbation in protein conformation. Nevertheless, the nucleotide-binding measurements do not support a specific interaction of Asp203 with the Mg2+ moiety of MgGTP.

/3-Tubulin

GTP-binding

(vi) 6>224X This mutation drasticallv diminished the requirement of Mg 2+ for GTP binding (by more than 2 orders of magnitude) without significantly altering GTP binding in the absence of Mg2+. GDP binding ( fMg2+) was relatively unaffected (Tables 3 and 4). The different results for the di- and triphosphate forms of the nucleotide can be rationalized by the fact that Mg2+ is required for GTP but not GDP-binding (Huang et al., 1985a; Correia et al., 1987; Farr et al., 1990). Mutation D224N therefore seemed to have specifically perturbed the /3, y-phosphate-binding region, i.e. the site of interaction of the magnesium moiety of MgGTP. Furthermore, mutations of conserved aspartic acid residues (D209N and D295N) on either side of Asp224 had relatively httle effect on nucleotide binding. These results are consistent with the hypothesis that Asp224 is either directly bound to the Mg2+ of MgGTP or is located in close proximity to this moiety. D224N was introduced into a sequence homologous to the Mg2+ binding site consensus sequence proposed by Burke et al. (1990) for ATP binding proteins (Fig. 6(b)). The finding that this mutation affects the Mg2+ site suggests that tubulin is evolutionarily related to the ATP-binding proteins (Ponstingl et al., 1981aJ; Krauhs et al., 1981) even though it binds guanine nucleotides with high specificity. Further studies will be required to substantiate this hypothesis.

5. Conclusion A detailed investigation of eight mutations in /?-tubulin’s GTP binding domain was initiated. This study demonstrated that several sequences identified on the basis of homology with the GTP-binding proteins do not participate as predicted in nucleotide binding to P-tubulin (Sternlicht et al., 1987; Fig. 6(a)). Conversely, our study suggests that a sequence identified on the basis of homology with ATP-binding proteins does participate in nucleotide binding. These results argue that tubulin belongs to a distinct class of GTP-binding protein unlike those corresponding to EF-Tu and ras protein (Dever et al., 1987). How large this class is and whether it is limited to the tubulin family is not known. We thank M. Yaffe for his compilation of sequence comparisons Fig. 6(b)) and his critical review of the manuscript. We are indebted to M. J. Short for his assistance with site-directed mutagenesis. We also thank B. Merrick, M. Maguire and J. Mieyal for helpful comments. We especially thank M. Burke for discussions concerning myosin structure. We are also indebted to the Natural Products Branch of the National Cancer Institute for the taxol used in this study. This work was supported in part by American Cancer Society grant CD-228K (to H.S.), National Institute of Health Pharmacological Sciences Training grant GM0 7382-11 (to G.W.F.) and grant P30CA43703 (to the Case Western Reserve University Cancer Center).

319

Domain

References Abbott, W. M. & Feizi, T. (1991). Soluble 14-kDa betagalactoside-specific bovine leetin. Evidence from mutagenesis by proteolysis that almost the complete polypeptide chain is necessary for integrity of the carbohydrate recognition domain. J. PIziol. Chem. 266, 5552-5557. Barahona, I., Soares, H., Cyrne, L.. penyue, D., i=. (1988). Denoulet , P. & Rodrigues-Ousada, Sequence of one u and two /?-tubulin genes of Tetrahymena pyriformis: structural and functional relationships with other eukaryotic tubulin genes.

J. Mol. Biol. 202, 365-382. Becker, A. B. & Roth, R. A. (1992). An unusual active site identified in a family of zinc metalloendopeptidases.

Proc. Nat. Acad. Sci., U.S.A. 89, 3835-383’9. Brown, H. R. & Erickson, H. P. (1983). Assembly of proteolytically cleaved tubulin. Arch. Biochem. Biophys. 220, 46651. Burke, D., Gasdaska, P. & Hartwell, L. (1989) Dominant effects of tubulin overexpression in Saccharomyces cerevisiae. Mol. Cell. Biol. 9, 104991059. Burke, M., Rajasekharan, K. N., Maruta, S. & Ikebe, M. (1990). A second consensus sequence of ATP-requiring proteins residues in the 21-KDa C-terminal segment of myosin subfragment 1. FERS Letters, 262, 1855188. Caplow, M. & Shanks, J. (1990). Mechanism of the microtubule GTPase reaction. J. Biol. Chem. 265, R9358941. Carlier, M.-F., Didry, D., Simon, 6. & Pantaloni, D. (1989). Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GTP-Pi using phosphate analogues. Biochemistry, 28, 1783-1791. Chiang, H. L. & Dice, J. F. (1988). Peptide sequences that target proteins for enhanced degradation during serum withdrawal. J. Biol. Chem. 263, 6797-6805. Clark, B. F., Kjeldgaard, M., la Cour, T. F., Thirup. S. & Nyborg, J. (1990). Structural determination of the functional sites of E. coli elongation factor Tu.

Biochim. Biophys. Acta, 1050, 203-208. Cleveland, D. W., Kirschner, M. W. & Cowan, N. J. (1978). Isolation of separate mRNAs from c(- and fi-tubulin and characterization of the corresponding in vitro translation products. CeEl, 15, 1021-1031. Correia, J. J., Baty, L. T. C Williams, R. C., Jr (1987). Mg2+ dependence of guanine nucleotide binding to tubulin. J. Biol. Chem. 262, 17278817284. Dalman, F. C., Scherrer, L. C., Taylor, L. P.. Akil, H. & Pratt, W. B. (1991). Localization of the 90-kDa, heat shock protein-binding site within the hormonebinding domain of the glucocorticoid receptor by J. Biol. Chem. 266, 3482-3490. peptide competition. Dever, T. E., Glynias, M. J. & Merrick, W. C. (1987). GTP-binding domain: three consensus sequence elements with distinct spacing. Proc. Nat. Acad. Sci., U.S.A. 84, 1814-1818. Farr, G. W., Yaffe, M. B. & Sternlicht, II. (1990). Alpha-tubulin influences nucleotide binding to betatubulin: an assay using picomoles of unpurified protein. Proc. Nat. Acad. Sci., U.S.A. 87, X%&5045. Fishback, J. L. & Yarbrough, L. R. (1984). Interaction of 6-mercapto-GTP with bovine brain tubulin. Equilibrium aspects. J. Biol. Chem. 259, 1968-1973. Fry, D. C., Kuby, S. A. & Mildvan, A. S. (1985). NMR studies of the MgATP binding site of adenylate kinase and of a 45.residue peptide fragment of the enzyme. Biochemistry, 24, 4680-4694.

6.

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Edited by D. DeRosier