Getting to the heart of β-tubulin

REVIEWS

The GTPases are a large group of functionally diverse enzymes involved in a variety of cellular events, including signal transduction, protein synthesis, mitosis and intracellular transportr. A common characteristic of these proteins, despite their divergent cellular roles, is that they act as binary on-off switches in which hydrolysis of bound GTP effects the transition between the on and off states2-4.A clear example of this is seen with ~21 + the GTP-bound state activates an intracellular signalling cascade, whereas hydrolysis of the GTP to GDP blocks signal transmissior?Jj. This principle also applies to P-tubulin (Box 1): the GTPbound form has a higher affinity for microtubule ends and promotes microtubule stabilization and growth, corresponding to an on signal, whereas the GDPbound form favours microtubule disassembly7J8. The importance of characterizing the GTP site of l3-tubulin is underscored by the relationship between GTP hydrolysis and the consequential treadmilling and dynamic instability of microtubules in vitro (see Box 1) and by the key role of these properties in the diverse behaviours of microtubules in vivo9.Furthermore, various medically and agriculturally important antimitotic drugs, such as the anticancer alkaloids from Vinca (vinblastine and vincristine) and Taxus (taxol), and the benzimidazole class of antifungal agents that bind to the colchicine site on tubulin, perturb these kinetic properties and the changes induced by the assembly-dependent GTP hydrolysislO. A full understanding of the molecular basis of microtubule assembly will therefore require high-resolution crystallographic structures of p-tubulin, both before and after GTP hydrolysis. Frustratingly, these structures, despite many attempts, have yet to be determined (see Box 2), and this has not only hindered a rational analysis of the tubulin GTP site, but also the full exploitation of tubulin-based therapies that target activity of the p-tubulin GTP site. An alternative approach to understanding the p-tubulin GTP site might be to compare p-tubulin with other GTPaseswhose crystallographic structures are known, since there is widespread evidence that catalytic mechanisms can be highly conserved. Protein crystallography has established unambiguously that the GTP-binding site is defined for several GTPasesuperfamily proteins (e.g. p21rus, elongation factor EF-Tu and transducin) by the peptides GxxxxGK, DxxG and NKxD, in the order specifiedil-r3 (Fig. 1; see Fig. 1 and its legend for details of peptide nomenclature used in this article). These consensus peptides have become an important tool for indicating whether novel proteins might also possess GTPase activity. Again, however, this approach has been frustrated when applied to p-tubulin since, despite being a bona fide GTPase, its primary sequence lacks the consensus peptides of the ‘conventional’ GTPasesuperfamily members. Nevertheless, this has led to a working hypothesis that the GTP-binding site of p-tubulin may resemble that of the various conventional GTPases. Some workers have taken the unusual liberty of allowing the reversal of some of the conventional GTPase consensus peptides (Fig. 1). Himan Sternlicht and co-workers proposed that the P-tubulin (103)KG trends in CELL BIOLOGY (Voi. 6) August 1996

Getting to the heart of p-tubulin Cellular microtubules assembleand disassembleat a variety of rates and frequencies,and theseproperties contribute directly to the cell-cycle-associatedrearrangementsof the microtubule cytoskeletonand to the molecular basis of mitosis. The kinetics of assembly/disassemblyare governed, in part, by the hydrolysis of GTP bound to the P-tubulin nucleotide-binding site. The p-tubulin GTP-binding site, therefore, lies at the heart of microtubule assembly-disassembly kinetics, and the elucidation of its structure is central to an understanding of the cellular behaviour of microtubules. Unfortunately, the crystallographic structure of p-tubulin is not yet available. In this review, we describethe progressbeing made using mutagenesisand biochemical studies to understand the structure of this unusual GTP-binding site.

HYTEG(~O~) sequence is equivalent to the GxxxxGK

consensus motif of conventional superfamily members, except that it is reversed, that the p-tubulin (295)D~KN(298) is equivalent to the superfamily NKxD, again in reverse orientation, and that the sequence (203)D~EA(206) is a conservative homologue of the superfamily DxxG (Ref. 14). Such sequence reversals might be considered unlikely; however, the HEXXH motif is found in both orientations in the active sites of zinc metalloendopeptidases?. A number of other bona fide GTPases, including the bacterial septum protein FtsZ (Ref. 16), the transglutaminase G,h (Ref. 17) and the chloroplast outer membrane protein IAP86 (Ref. 18), also lack the consensus peptides characteristic of the conventional members of the GTPasesuperfamily. A further complication is that the primary sequences of certain of these atypical GTPases contain a highly conserved glycine-rich peptide that is characteristic of many ATPases and interacts with the phosphates of the bound adenine nucleotide. The highly conserved glycine-rich peptide is present in both p-tubulin [(140)GGGTGSG(146); Ref. 141 and in another atypical GTPase, FtsZ (GGGTGTG; Ref. 19; Fig. 1). Currently, it is unclear whether these atypical GTPases, including the tubulins, represent parallel solutions to the GTP-binding problem. Alternatively, the precise conjunctions of active-site groups may be the same in all the GTPases, conventional and atypical: Lawrence Pearl has noted that p-lactamase and endothiapepsin lack significant sequence homology, yet have related catalytic mechanisms and similar conjunctions of active-site groupszO. 0 1996 Elsevier Science Ltd

PII: SO962-8924(96)10024-6

Roy Burns is in the Biophysics

SectionB,ackett Lahora;ory Imperial CAllege

of Science

~~~.?~~g~o~~on UK SW7 ;BZ; and Kevin Farrell is in the Dept of

Mo,ecu,arCe,,u,ar

and Developmental Biology, University

of Ca,ifornia SantaBarb’s CA 93106, USA. ’

297

Testing whether specific the GTP site of p-tubulin

peptides

contribute

to

A classic approach for testing whether specific peptides contribute to active or ligand-binding sites of proteins has been to examine the biochemical effects of sequence changes introduced by site-directed mutagenesis. This approach is feasible for the P-tubulin GTP site if the mutated p-tubulin is isolated either from mutant yeast strains or is expressed in vitro (see Box 2). Such mutagenesis studies mtist, in the absence of a crystallographic structure for tubulin, be interpreted with caution since the loss of GTP binding or hydrolysis activities could simply be due to remote effects unrelated to the GTP site. The interpretation of such studies can be augmented by considering whether the target residue(s) is highly conserved. This is particularly powerful when considering the 298

mutagenesis of P-tubulin since a large number of sequences are available from phylogenetically diverse organisms, and peptides that are highly conserved across all phyla would be predicted to be functionally important. The (103)KG~y~~G(109) p-tubulin peptide, which has been implicated as a reversed homologue of the GxxxxGK motif of the superfamily GTPases14, is highly conserved 21. This implies that it is functionally important, and the mutagenesis studies are consistent with it playing a role in the assemblydependent hydrolysis of GTP. An E108A-ElllA double mutation in the Saccharomycescerevisiaegene encoding p-tubulin, TUBZ, was viable in haploids but blocked cells immediately prior to mitosis upon transfer to a restrictive temperaturez2. Other TUB2 Thrl07 mutations (T107G, T107K and T107W) were haploid lethal, but tubulins isolated from viable heterozygous diploids increased both the assemblydependent rate of GTP hydrolysis by 4-lo-fold, and increased the steady-state critical concentrationz3. These mutations also suppressed the growing and shortening rates of microtubules in vitro and caused a mitotic slowdown in the heterozygous cellsz4. The biochemical and phenotypic effects of mutations within the (103)KG~~‘r~G(109) peptide are consistent, therefore, with this peptide playing a role in GTP hydrolysis, and suggest that one effect of the mutations is to increase the probability of loss of tubulin subunits from microtubule ends following tubulin-GTP hydrolysis. This would account both for the apparently greater rate of GTP hydrolysis of the mutated subunits, as well as for the slower growth rate of mutated microtubules in vitro and slower mitosis in cellsz4. There are, however, apparent differences between this peptide and the superfamily GxxxxGK motif. For example, LyslO3 is conserved in 148 out of 150 available @ -tubulins, yet a K103M mutation was haploidviable, and mutated microtubules assembled in vitro have an assembly-dependent rate of GTP hydrolysis indistinguishable from that of wild-type microtubulesz3. Similarly, Gly109 is highly conserved in 149 out of 150 p-tubtilin sequences, yet a G109E mutation in the Caenorhabditis elegans met-7 p-tubulin gene had only a semi-dominant phenotypeZ5. By contrast, corresponding mutations in the GxxxxGK motifs of superfamily members affect both GTPbinding and hydrolysis26,27.However, even in the consensus motifs of conventional GTPases, mutations can have unpredictable effects. For example, a GlZV mutation in the (~O)GAGGVGK(~~)peptide of p21”” reduced GTPase activity, yet valine is normally present at the equivalent position (Va120; Ref. 28) in wildtype EF-Tu, which is another conventional GTPase. Such unexpected differences probably reflect both our current lack of understanding of protein structure and subtle differences between the catalytic mechanisms of conventional GTPases. In this context, if the (~O~)KGHYTEG(~O~)p-tubulin peptide is homologous to the superfamily motif, there must indeed be subtle differences in the catalytic mechanisms between p-tubulin and the conventional GTPases. trends in CELL BIOLOGY (Vol. 6) August 1996

That there may be a potential difference is reinforced by the mutagenesis of the (295)DAKN(298) and (203)D~~A(206) B-tubulin peptides, which are putative homologues of the superfamily NKxD and DxxG motifs. If the B-tubulin (295)DAKN(298) peptide is equivalent to the NKxD motif, it should interact with the purine base of the bound nucleotide. In particular, evidence from the mutagenesis of ~21”~ and EF-Tu indicates that a D295N mutation should change the base-binding specificity of B-tubulin from GTP to XTP. However, this was not observed with mutated tubulin isolated from Saccharomyces cerevisiae2gor with chick tubulin expressed in vitro30. Moreover, the conventional GTPase paradigm predicts that tubulins carrying the N298K and N298Q mutations should have greatly reduced affinities for GTP, but again the measured GTP-binding affinities of these altered B-tubulins were indistinguishable from that of wild-type tubulin29. Furthermore, a D295A-K297A double mutation in Saccharomyces cerevisiaeB-tubulin also had little effect on cells apart from a slightly enhanced sensitivity to benomylz2. The DaKN peptide is unlikely, therefore, to be functionally equivalent to the superfamily NKxD motif. Turning to the (203)D~~A(206) peptide, analysis of the conventional superfamily GTPases leads to the prediction that mutations of the Asp203 (which is conserved in 172 out of 173 B-tubulin sequences) would reduce Mg2+-GTP binding by interfering with the phosphate and magnesium coordination. Tubulin purified from a yeast mutant heterozygous for a D203S mutation, however, had unaltered GTP binding. Furthermore, the assembly-dependent rate of GTP hydrolysis, the kinetics of microtubule growing and shortening and the cell growth rate were not significantly affected2g. By contrast, GTP and GDP binding to chick p-tubulin expressed in vitro were markedly affected by a D203N mutation, but the protein was folded anomalously and its ability to coassemble with exogenous brain tubulin was impaired30. It is unlikely, therefore, that the B-tubulin DNEA peptide coordinates the bound nucleotide. The high degree of conservation of this site and the increased coldand benomyl-sensitivity of a double D203A-E205A yeast mutant22 do, however, imply that this sequence is both functionally important and highly susceptible to the conformational state of the protein. Although the (140)GGGTGSG(146) B-tubulin peptide exhibits a homology to sequences common to various ATPasesrather than the conventional GTPases (but see below for further discussion of this point), there is increasing evidence that it contributes to the B-tubulin GTP-binding site. The peptide is common to 135 of 171 p-tubulin sequences (with a conservative S145A substitution in 34 of the exceptions) and its potential importance is indicated by the severe recessive phenotype of a G141E mutation in Caenorhabditis elegans25.A T143G mutation in yeast p-tubulin reduced both GTP binding and hydrolysis and suppressed the growing and shortening rates of microtubules in vitro (C. A. Dougherty and K. W. Farrell, pers. commun.). This is significant since equivalent mutations of adenylate kinase31,32and FtsZ16s33 also reduce the nucleotide-binding and catalytic activities. trendsin CELL BIOLOGY (Vol. 6) August 1996

BOX 2 - WHY 6 THERE NOT YET A HIGH-BESOLUTIO~ p-TUBlJLf N? ---A high-resolution structure of the tubufin heterodimer has yet to be obtained, although the structure of Zn2*-induced tubufin sheets has been analysed to 6.54 resofution by image analysis of low-dose electron micrographssg. Three factors have probably contributed to the difficulty in crystaflizing tubulin. First, the protein purified from most sources is a mixture of genetically defined o(- and p-isoforms, wfth further heterogeneity originating with the multiple post-translational modifications. Second, tubufin exhibits a structural plasticity as demonstrated by its intrinsic lability and by the differing kinetic properties of tubulin subunits complexed with CTP or GDP (see Box 1). Finally, tub&in has an inherent propensity to assemble into microtubules and other structural pofymorphs; the subunit-subunit interactions involved in forming these structures may be kinetically more favourabfe than those required for crystal growth. The obvious approach to eliminate the genetic and post-translational heterogeneity would be to express genes encoding specific OL-or f3-tubulins in Escherichiaco/i. Unfortunately, the functional folding of the nascent tub&in polypeptides has an absolute requirement for the mu&subunit chaperonfn TCPI (Refs 60 and 61), which restricts any functional expression of tub&in to eukaryotic systems. A further complication is that there is increasing evidence that additional cofactors are required for the functional folding of a- and p-tubuiins62x63. For the future, some of the other atypicaf CTPases may prove more amenable for high-resolution crystallographic studies (e.g. FM), which in turn could facilitate the derivation of working models for the structures of the tubutin family of GTP-binding proteins. The absence of a high-resolution tubulin structure has precluded a rational approach to the elucidation not merely of the tubulin GTP sites but also of the tubutin sites to which medically and agriculturally important drugs bind, and which presumably usurp the normal functions of the natural microtubule regulators that are assumed to bind to the tubulin drug sites. Only two approaches are currently available that allow the biochemical properties of mutated tubulins to be examined. First, genes encodfng specific tubulins can be expressed in vitro using a reticufocyte or wheat germ expression system34 but this approach yields in the order of nanogram amounts of tubulin and precludes direct assay of the assembly and GTP-binding properties of the expressed protein. Alternatively, milligram amounts of Saccharomyces cerevisiae tubufin can be purified following large-scale fermentations, and this has allowed biochemical analysis of specific mutations in the single Btubulin (TUB2) gene64.

Crosslinking

GTP to P-tubulin

The mutagenesis studies have so far failed to identify potential p-tubulin peptides that interact with the guanine base, but several candidate sequences have been implicated by GTP-crosslinking methods. This approach has the potential for identifying peptides that lie close to, or interact with, the guaninenucleotide base since irradiation of the tubulin-GTP complex causes radical formation, which attacks the purine ring and results in its covalent attachment to the protein. Unfortunately, the crosslinking results have not provided a coherent picture, unlike the situation with conventional GTPases where periodate-induced crosslinking of GTP to p21rm, EF-Tu and T-cell receptor y-chain correctly identified the NKxD consensus motif34-36. The different B-tubulin sequences identified by the crosslinking studies may in part derive from perturbation of the tubulin structure, which exhibits considerable conformational plasticity (see Box l), by the highenergy irradiation necessary to induce covalent ring attachment. 299

1

(103)i&tiME~(1-0~) I

1

I

1

(203)DN~iX(207) I I

/I-

383

FIGURE 1 Comparison of the GTP-binding motifs of p21 rclswith those proposed for the atypical GTPases, B-tubulin and FtsZ. The principal peptides of human p21 rOSthat interact with the guanine or phosphate groups (P/Mg) are shown, together with the putative equivalents for B-tubulin and Escherichia co/i FtsZ. Note the sequence reversals of the putative p-tubulin consensus motifs14. The residue numbers in this figure and throughout the review relate to the human Bl -tubulin isoform; small differences will occur with other B-tubulin sequences owing to a limited number of insertions and deletions. In addition, the sequences of the specified peptides will differ in the B-tubulins from other organisms; the altered residues are almost invariably at the positions where there is a divergence between the human Bl sequence and that of the conventional CTPases. Large capitals signify consensus residues and the remainder are displayed as small capitals. In the text, sequences that do not have a consensus homologue are given in large capitals.

The P-tubulin:63-77 peptide has been labelled via the base using both unmodified GTP and an S-azidoGTP analogue37,38. Surprisingly, peptide 65-79 has also been identified by using a GTP derivative with the reactive group coupled to the r-phosphate39. The functional importance of a peptide common to these sequences [(Gl)PRAILVDLEP(70)] is indicated by its remarkable conservation in almost all P-tubulins, yet the available mutagenesis data fail to indicate a role for this peptide in nucleotide binding. A P61L or a P61S mutation has a semi-dominant phenotype in Caenorhabditis elegansZ5,whereas a D67A-E69A double mutation in Saccharomycescerevisiae has no apparent effectz2. Site-directed mutagenesis of the chick gene also failed to indicate a role for Asp67 or Glu69 in the p-tubulin GTP-binding site30, although there is some question as to the validity of the method used to measure GTP binding in this studyz9. An important caveat, however, is the uncertainty of the phenotype of mutations affecting nucleotide binding - for example, a yeast T143G p-tubulin mutation reduced GTP binding at least tenfold in vitro yet was viable in haploid cells (C. A. Dougherty and K. W. Farrell, pers. commun.). The (Gl)PRAILVDLEP(70) sequence cannot be excluded, therefore, as contributing to the GTP site, particularly as the Asp67 is very highly conserved (145 out of 146 300

P-tubulins), and conserved aspartates play key roles in nucleotide binding in conventional GTPases’“, ATPases40and a GTP cyclohydrase41. The P-tubulin:l%-174 peptide also has been labelled with unmodified GTP, and an antibody against part of this peptide inhibits GTP binding42J43. Mutations in the conserved charged amino acids in this peptide (Arg156, Glu157, Glu158, Aspl61, Arg162 and Lys174) also result in a variety of recessive-lethal, benomyl-supersensitive and cold-sensitive phenotypes 22.However, this p-tubulin: 155-174 peptide is not very highly conserved. Therefore, it is unlikely to contribute directly to the GTP-binding site. The most persuasive photoaffinity labelling relates to the P-tubulin:3-19 peptide. It has been identified using both unmodified GTP (Ref. 44) and 8-azidoGTP (Ref. 45). There is direct evidence that the guanine base can be covalently coupled to Cysl2 (Ref. 44), a residue that is conserved in 149 out of 151 p-tubulin sequences. Furthermore, Cysl2 can be crosslinked to either Cys201 or Cys211 by N’N’ethylene-bis(iodoacetamide) provided that the P-tubulin E-site is first depleted of the bound nucleotide46. These results strongly suggest that the Cysl2 lies close to the GTPbinding site. In summary, mutational and crosslinking analysis of the p-tubulin GTP site indicate that both the trends in CELL BIOLOGY (Vol. 6) August 1996

SQAGQCGNQIGl7

61PRAILVDLEB70

hvGQaGvQIG QlGQCGNQIG hvGQCGNQla QAGQCGNhvG

PRAvfVDLEP PRAvLlDLEP PRAvLVDLEP PRAImmDsEP

140 G G G T G S G 146 GGGTGSG aGGTGSG sGGTGSG aGGTGSG

II

rGWYTiG sG.fsqG sG.YcqG nG.YdiG

155IREEYPDRIMNTFSVVPSPRl74 m

178 TV

1svdYgkkskleFSiyPaPq 1ndrYPkklvqTySVfPnqd 1REafPkkviqTySVfansd IcdrYPkkIltTySVfPar.

II

aVVEPYN

I

-

vVVQPYN

vVVhPYN vVVqsYN

200YCIDNEALYDICFRTLRLTTPTYGDL225 fmvDNEAiYDICrRnLdierPTYtnL vvlDNtALnrIatdrLhiqnPsfsqi vvlDNaALhrlssgkfktdTPTfdhi vvfDNasLlnIsgkvfRnpnidlqht

V E P Y N 184

295 I) AK

240 L R F P G 244 m

LRFdG LRyPG YRFi2S iRFPs

m

a 298

epat4 QPKN kpts DpsN

FlGURE 2 Alignment of the nine peptides of the c1-to e-tubulins 48 implicated as contributing to the CTP-binding site. The upper line lists the B-tubulin sequences, and the four other lines the 01-,y-, 6- and e-tubulin sequences. The 01-, B- and y-tubulin peptides correspond to the human sequences; 6-tubulin is from Caenorhabditis elegans; and l -tubulin is from Saccharomyces cerevisiae48. Residues that are common to the B-tubulin sequences are shown in capitals. The peptides 8-Tu:8-17, B-Tu:61-70, 8-Tu:155-174, B-Tu:178-184 and B-Tu:295-298 have been implicated in base-binding; B-Tu:103-109 and p-Tu:l40-146 in phosphate coordination; p-Tu:200-225 in Mg2+ binding; and B-Tu:240-244 in ribose binding. The B-tubulin (295)D~ti(298) sequence lies within p-Tu:200-225. The lack of conservation between the six tubulin families strongly suggests (see text) that certain of these peptides do not contribute to the GTP-binding site.

(103)KGHYTEG(109) and the (140)GGGTGSG(146) peptides are important for B-tubulin GTP hydrolysis and that residues flanking Cysl2 contribute to the base binding. By contrast, the available evidence fails to support the proposition that the (203)D~~A(206) and the (295)DAKN(298) peptides play a role in the p-tubulin GTP site, and this indicates that the Btubulin GTP site is structurally distinct from that of the conventional GTPases.It is clear, therefore, that there are limitations to any analyses based upon sequence comparisons and that great caution must be applied to any interpretation.

Mg2+, particularly since either Cys201 or Cys211 can be crosslinked by N’N’ethylene-his-(iodoacetamide) to Cysl2 (Ref. 46), and crosslinking shows that this residue lies close to the bound guanine. Two general points emerge from these studies. First, all of the identified peptides lie within the N-terminal half of the p-tubulin sequence. This supports a two-domain model for the structure of the tubulin dimer, based upon an analysis of y-tubulin sequences, with the GTP-binding site being defined by residues of the N-terminal domain47. Second, none of the B-tubulin peptides implicated in guanine-base binding shows clear homology with The tub&n family has a distinctive CTP-binding other GTPases,either conventional or atypical, while site only a limited homology exists for B-tubulin peptides While the experimental evidence points to key roles implicated in binding Mg2+/phosphate. B-Tubulin, for the (103)KGHYrEG(109)and (140)GGGTGSG(146) therefore, presents an unusual picture in that the peptides and the residues flanking Cysl2, contribupeptides implicated in the GTP-binding pocket are tions by other peptides to the B-tubulin GTP site are a mixture of sequences unique to B-tubulin, and not ruled out, and indeed B-tubulin may share adsequences common to both GTPasesand ATPases. ditional features with other nucleotide-binding proPeptides contributing to the B-tubulin GTP site teins. In particular, sequence homologies have been might also be expected to be present in other memnoted between B-tubulin sequences (178)TWE(181) bers of the tubulin family (a-, y-, S- and E-) since the and (240)LRFPG(244) and peptides associated with family members share 3540% sequence identity47,48. base-and ribose-binding activities of various ATPases37. Comparison of the putative GTP-binding peptides, Both these B-tubulin peptides are highly conserved however, reveals a gradation, ranging from peptides (163 out of 173, and 16.5 out of 175, respectively, that are conserved in all members of the tubulin famare absolutely conserved), and mutations in these ily to others that exhibit member-specific divergence sequences result in strong phenotypeszzJ5. The (Fig. 2). This presents a conundrum: do the memberp-tubulin sequence (ZOO)YCIDNEALYDICFRTLRLTPTspecific variants provide evidence that these peptides yGD~(2.25) reportedly shows some homology with are not involved in defining the GTP site or do they the Mg2+-binding sequence RCNGVLEGIRIC.RKG.FPNRILreflect the differing biochemical and functional properYGDF of myosin and other ATPases30.Parts of the ties of the tubulins? For example, 01-and B-tubulins P-tubulin:200-225 peptide may possibly lie close to both bind GTP, yet only B-tubulin hydrolyses bound the bound nucleotide and coordinate the bound GTP upon assembly into microtubules (Box 1). By trends in CELL BIOLOGY (Vol. 6) August 1996

301

contrast, y-tubulin does not polymerize into microtubules, is located primarily in microtubuleorganizing centres, and its nucleotide-binding properties (together with those of the S- and e-tubulins) are unknown47. The varied conservation is illustrated by the retention in all tubulin family members of homologues of the p-tubulin (140)GGGTGSG(146), (61)PRAILVDLEP(70), (178)TVVE(181) and (240) LRFPG(244) peptides and the base-binding peptide flanking B-tubulin Cysl2 (Fig. 2). By contrast, the putative magnesium-binding sequence of p-tubulin [(200).C....IC.R....YGD.(225)] shows significant differences in the other tubulins (Fig. 2). The most marked differences, however, relate to the p-tubulin (~O~)KGHYTEG(~O~) peptide, implicated by the mutagenesis studies in contributing to the catalytic mechanism 23. This peptide is absent from the y-, S- and e-tubulins (Fig. 2) and is altered to RGHYT(I/v)G in 138 out of 149 of the available ol-tubulin sequences.

The future

Such observations highlight the importance of determining the high-resolution structure of the tubulin subunit. The mutagenesis studies, subject to their limitations, indicate that p-tubulin probably has a GTP-binding site that is significantly different from that of the GTPase-superfamily proteins. Similarly, sequence analysis reveals that the homology between p-tubulin and various ATPases is extremely limited. Both approaches rely on primary sequence analysis. They may, therefore, both fail to detect a more fundamental similarity since there is an increasing number of examples of proteins that share a common tertiary structure yet have markedly different primary sequences (e.g. actin, hsp70, hexokinase). B-Tubulin may share a tertiary fold with one or more proteins, yet this will probably only become apparent once its crystallographic structure becomes available. One intriguing possibility is that B-tubulin may have a similar fold to that of actin, even though the two proteins have strikingly different primary sequences. Both The glycine-rich sequences proteins, however, self-assemble, hydrolyse a bound Some authors have equated the GxxxxGK convennucleotide triphosphate, the GTP- (or ATP)-cap tional GTPase motif with the glycine-rich sequences model (see Box 1) applies to both microtubules and often found in ATPases (e.g. Ref. 49), partly because microfllaments8, and both proteins require the TCPl chaperonin (see Box 2) for their folding into a funcof extensive evidence that both contribute structional form. Alternatively, the tertiary structure of turally to the coordination of bound phosphates p-tubulin may resemble another nucleoside triphosand partly because the GxxxxGK motif is found in the glycine-rich sequences of some ATPases [e.g. phatase, may contain elements from a variety of (~.S)GGPGSGKG(~~)of adenylate kinases]. However, different proteins (as indicated from the current the p-tubulin mutagenesis evidence implicating primary sequence analysis) or may be unique. both (140)GGGTGSG(146) and (~O~)KGHYTEG(~O~) A further area that is likely to evoke considerable interest is the role of or-tubulin. Analysis of the priin phosphate coordination and the fact that the GxxxxGK motifs of some conventional GTPases are mary sequence indicates that it contains homologues not particularly glycine-rich [e.g. (~~)GHvDHGK(~~) of the peptides implicated in defining the B-tubulin GTP-binding site, and the subtle residue differences of Escherichia coli EF-Tu] suggest that these two motifs (e.g. RGHYTIGvs. KGHYTEG)may account for the difmay not be interchangeable. Two features characterize the GxxxxGK motif of the conventional GTPases fering enzymatic activities (see Box 1). Surprisingly, and the glycine-rich peptides of various ATPases. little is understood about why microtubules are assembled from LX-Bheterodimers, and this is partly beFirst, the available crystallographic evidence demonstrates that they have related tertiary structures (see cause most mutations affecting ligand sensitivity Ref. SO). Second, although the peptides are strongly map to the gene encoding p-tubulin. A possible clue to the c&ubulin function comes from the activation conserved within related proteins, only the diagnosby accessory proteins, such as the GTPase-activating tic residues are fully conserved, and mutations within proteins (GAPS), of the hydrolysis reaction of memthe motifs of different proteins can result in differing bers of the GTPase superfamilyl. The p-tubulin effectsso. Consequently, there are subtle differences GTPase activity is greatly stimulated following oc-B in phosphate coordination between different prodimer addition to the microtubule end. This actiteins; the inverted (103)KG~~r~G(109) peptide of vation, therefore, appears to result from the interp-tubulin may simply be a more extreme variant action between a p-monomer at the microtubule end of this heterogeneity. This idea is supported by the and the incoming a--B heterodimer, and this implies equivalent a-tubulin peptide: RGHYTIGK (Fig. 2). It that the assembled a-monomer may activate this can be considered to be a homologue of the inverted hydrolysis. Furthermore, the question of whether p-tubulin (~O~)KGHYTEG(~O~)peptide, a homologue a-tubulin can exist in two or more states (equivalent of the GxxxxGK motif and an inverted homologue to the GTP- and GDP-states of p-tubulin) remains of the GTGSFGR peptide that forms the phosphateunresolved, together with the role of the nonbinding loop of many protein kinases. The tubulin exchangeable GTP. sequences, therefore, reinforce the conclusion that It is now 25 years since microtubules were first asproteins have evolved a variety of ways for effectsembled in vitro and the importance was ascertained ing phosphate binding. Indeed, the presence of both (140)GGGTGSG(146) and (~O~)KGHYTEG(~W) of including GTP in the assembly buffer. Although many aspects of the assembly kinetics have since peptides in p-tubulin (and their homologues in been elucidated, we still do not understand fully the a-tubulin) may reflect unique requirements for phosmechanism by which the GTP hydrolysis alters the phate coordination related to the functional roles of properties of p-tubulin, the role of the a-subunit or tubulins in microtubules. 302

trends in CELL BIOLOGY (Vol. 6) August 1996

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