Refined Structure of (alpha)(beta)-Tubulin at 3.5 A Resolution tubulin...improved by incorporating 114 additional diffrac-tion patterns. A few of the patterns extend to 2.5 A˚ . However

$Page 1: Refined Structure of (alpha)(beta)-Tubulin at 3.5 A Resolution tubulin...improved by incorporating 114 additional diffrac-tion patterns. A few of the patterns extend to 2.5 A˚ . However$
doi:10.1006/jmbi.2001.5077 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 313, 1045±1057

Refined Structure of aaabbb-Tubulin at 3.5 AÊ Resolution

J. LoÈ we1, H. Li2, K. H. Downing2 and E. Nogales2,3*

1MRC Laboratory of MolecularBiology, Cambridge, UK2Life Science Division,Lawrence Berkeley Natl. Lab.Berkeley, CA 94720USA3Howard Hughes MedicalInstitute, UC BerkeleyBerkeley, CA 94720-3200USA

E-mail address of the [email protected]

0022-2836/01/051045±13 $35.00/0

We present a re®ned model of the ab-tubulin dimer to 3.5 AÊ resolution.An improved experimental density for the zinc-induced tubulin sheetswas obtained by adding 114 electron diffraction patterns at 40-60 � tiltand increasing the completeness of structure factor amplitudes to 84.7 %.The re®ned structure was obtained using maximum-likelihood includingphase information from experimental images, and simulated annealingCartesian re®nement to an R-factor of 23.2 and free R-factor of 29.7. Thecurrent model includes residues a:2-34, a:61-439, b:2-437, one molecule ofGTP, one of GDP, and one of taxol, as well as one magnesium ion at thenon-exchangeable nucleotide site, and one putative zinc ion near theM-loop in the a-tubulin subunit. The acidic C-terminal tails could not betraced accurately, neither could the N-terminal loop including residues35-60 in the a-subunit. There are no major changes in the overall fold oftubulin with respect to the previous structure, testifying to the quality ofthe initial experimental phases. The overall geometry of the model is,however, greatly improved, and the position of side-chains, especiallythose of exposed polar/charged groups, is much better de®ned. Threeshort protein sequence frame shifts were detected with respect to thenon-re®ned structure. In light of the new model we discuss details of thetubulin structure such as nucleotide and taxol binding sites, lateral con-tacts in zinc-sheets, and the signi®cance of the location of highly con-served residues.

# 2001 Academic Press

Keywords: tubulin; taxol; zinc-sheets; electron crystallography;crystallographic re®nement
*Corresponding author
Introduction

Microtubules are cytoskeletal polymers essentialin all eukaryotic cells, with functions extendingfrom cellular transport to cell motility and mitosis.1

They are made of repeating ab-tubulin heterodi-mers that bind head to tail into proto®laments.About 13 of these proto®laments associate in paral-lel making the microtubule wall, and giving rise toa polymer with a well de®ned polarity. Essential tothe function of microtubules is their ability toswitch stochastically between growing and shrink-ing phases (dynamic instability),2 a non-equili-brium behavior of tubulin that is based onnucleotide binding and hydrolysis. Each tubulinmonomer binds one molecule of GTP. The nucleo-tide bound to a-tubulin, at the so called N-site, isnon-exchangeable. The nucleotide bound to b-tubulin, at the E-site, is exchangeable. GTP isrequired at the E-site in order for tubulin to poly-merize,3 but this nucleotide is hydrolyzed and

ing author:

becomes non-exchangeable upon polymerization.The resulting metastable microtubule structure isthought to be stabilized by a cap of remainingGTP-tubulin subunits at the ends, the loss of whichresults in rapid depolymerization.

The structure of the tubulin dimer was obtainedby electron crystallography of zinc-induced tubulinsheets,4 which are formed by the antiparallelassociation of proto®laments. Addition of taxolstabilized the sheets against cold temperaturedepolymerization and aging,5 similar to the effectsof taxol on microtubules. Using low-dose methods,cryo-preservation and image processing, a struc-ture of the tubulin dimer bound to taxol wasobtained at 3.7 AÊ resolution that included all butthe last acidic C-terminal residues. Each compactmonomer contains an N-terminal, nucleotide-bind-ing domain, comprising six parallel b-strands (S1-S6) alternating with helices (H1-H6). The loopsjoining each strand with the beginning of the nexthelix are directly involved in binding the nucleo-tide (loops T1-T6). Within each monomer, nucleo-tide binding is completed by interaction with the

# 2001 Academic Press

1046 Re®ned Structure of ��-Tubulin

N-terminal end of the core helix H7. The core helixconnects the nucleotide binding domain with thesmaller, second domain, formed by three helices(H8-H10) and a mixed beta sheet (S7-S10). The C-terminal region is formed by two antiparallelhelices (H11-H12) that cross over the previous twodomains. The N-site GTP in a-tubulin is buried atthe monomer-monomer interface within the dimer,while the GDP at the E-site is exposed on the sur-face of the dimer, thus explaining the exchangeabil-ity of the nucleotides.

The longitudinal contact between subunits, verysimilar between monomers within the dimer andbetween dimers, is very extensive (about 3000 AÊ 2

are buried with the formation of the dimer fromthe monomers, or in the contact betweendimers).6,7 The two surfaces involved in the inter-faces are convoluted in shape and highly comp-lementary. About 52 % of the residues at theintradimer interface are totally conserved acrossspecies, while about 40 % are conserved at theinterdimer contact.8 Upon polymerization the E-site nucleotide becomes buried at the newlyformed interface. Loop T7, a region in the tubulinstructure opposed to the nucleotide site, isinvolved in the interaction with the nucleotide inthe next subunit along the proto®lament.6 T7includes highly conserved residues in both tubulinsubunits (GXXNXD). This conservation extends totheir bacterial homologue FtsZ.6,9 The interactionwith the nucleotide across the longitudinal inter-face is completed by Lys254 in b-tubulin (withinthe H8 helix), which interacts with the g-phosphateof the N-site nucleotide, and in a-tubulin byGlu254, which is in a position that would be closeto the g-phosphate of the E-site nucleotide (in thecrystal structure this nucleotide is GDP afterhydrolysis during the formation of the sheets). Thecorresponding aspartic acid in FtsZ has beenshown to be required for nucleotide hydrolysis,10

supporting the idea that a: Glu254 is essential forthe polymerization-dependent hydrolysis. In yeastTUB1 mutation of Asp251 and Glu254 to alanineresidues results in a dominant lethal phenotype.11

Each mutation on its own is a dominant lethal, andtransient expression of the mutant a-tubulinsresults in hyperstable microtubules (K. Anders andD. Botstein, personal communication), in furtheragreement with the requirement of these residuesfor nucleotide hydrolysis.

A high resolution model of the microtubule wasobtained by docking the crystal structure of thetubulin proto®lament into a reconstruction of themicrotubule obtained by cryo-electron microscopyand helical image reconstruction.7 The plus end ofthe microtubule is crowned by b-tubulin subunitsexposing their nucleotide surface to the solution,while the minus end is crowned by a-subunitsexposing their catalytic end. This orientation hasvery important repercussions for the GTP-capmodel of microtubule dynamics.8

The docking showed that the C-terminal helicesform the crest of the proto®laments on the outside

surface of the microtubule. The bumpy inside sur-face of the microtubule is de®ned by a series ofloops: loops H1-S2 and H2-S3, which were poorlyresolved in the original crystal structure of thesheets; and the S9-S10 loop, which is eight residueslonger in a-tubulin and in b-tubulin forms part ofthe taxol binding site. The docking indicates thatthe lateral contact between proto®laments is domi-nated by the interaction of the M-loop, the loopbetween S7 and H9, with loop H1-S2 and helix H3.This interaction, in comparison with the longitudi-nal contact, has an important ionic contribution,both for a-a and b-b contacts. The M-loop is in aposition where it could hinge without disruptingits interaction with the adjacent subunit, therebyallowing for the known variability in proto®lamentnumber of reconstituted microtubules. Thesequence of this loop corresponds to one of themost divergent segments between a and b-tubu-lins. In b-tubulin the M-loop is an essential part ofthe taxol binding pocket, while H3 follows loopT3, which is involved in binding the g-phosphateof the E-site nucleotide. The conformation of theM-loop is stabilized in the a-subunit by the longS9-S10 loop. In the b-subunit a similar stabilizingfunction may be played by taxol and taxol-likecompounds.12 On the other hand, the destabilizingeffect of nucleotide hydrolysis may be due to aconformational change transmitted to H3 throughthe g-phosphate sensing loop.

Here we present a re®ned model of the ab-tubu-lin dimer to 3.5 AÊ resolution, carried out usingstandard X-ray crystallography methodology. Thenew structural model is very similar to that orig-inally published but has much improved geometry,better de®ned side-chain conformations andincludes three protein sequence frame shifts of oneresidue in both a and b-tubulins. Assignmentswere made for a magnesium ion in the non-exchangeable site, and a zinc ion at the lateral con-tact between a subunits. We discuss in detail thelateral contacts in zinc-sheets and the nucleotideand taxol binding sites.

Results

Previous to the re®nement the data set wasimproved by incorporating 114 additional diffrac-tion patterns. A few of the patterns extend to2.5 AÊ . However the completeness at this resolutionis very low. In the resolution shell from 3.7 to3.5 AÊ the completeness dropped to 83.7 %, whichwe considered the lower limit for this work. Theoverall I/(sI) for this data set was 5.4, 2.3 for thelast resolution shell ( 3.7-3.5 AÊ ). The overall multi-plicity was 6. No attempt has been made to correctfor multiple scattering and diffuse scattering aswas previously done for bacteriorhodopsin.13 Mul-tiple scattering has been shown to be very smallfor such thin crystals.14

The unre®ned model of the tubulin dimer (PDBID 1TUB) was ®rst re®ned as two rigid bodies

Re®ned Structure of ��-Tubulin 1047

which resulted in a crystallographic R-factor ofabout 42.0. Several cycles of maximum-likelihoodincorporating experimental phase information,simulated annealing, positional re®nement, con-strained temperature re®nement and manualrebuilding using O, greatly improved the phase-combined 2Fo ÿ Fc density and the R-factors (®nalR-factor: 23.2, Rfree: 29.7). Torsion angle re®nementdid not improve convergence signi®cantly, so clas-sical Cartesian re®nement was used. A major dropin R-factors was observed when anisotropic overalltemperature factors were used. This is due to theproblem of imperfect specimen ¯atness whichcauses amplitudes to fall off faster in the directionperpendicular to the crystal plane than parallel tothe plane 15 (temperature factors: B11 � 26.54 AÊ 2,B22 � 10.8 AÊ 2, B33 � ÿ 37.35 AÊ 2, the last corre-sponding to the direction perpendicular to thecrystal plane). Difference maps and 2Fo ÿ Fc mapswere calculated using Fcalc values in place of miss-ing Fobs in the missing cone, a necessary step thatintroduced some model bias.

A number of register errors in the starting modelwere removed in this process. A magnesium ionwas placed in the N-site in a position similar tothat of magnesium in other nucleotide binding pro-teins and the FtsZ:GMPCPP crystal structure (S. C.Cordell and J.L., unpublished results). A large

difference peak at the lateral interface was inter-preted as a zinc ion. Further improvement of theR-factors was achieved by careful optimization ofcell constants a and b. A procedure was used inwhich several cycles of rigid body re®nement andpositional minimization were performed for onedimensional scans of cell constants a and b, moni-toring Rfree. The clear minimum of this search wascon®rmed by greatly improved bond lengthaverages of the ®nal model when compared tostandards derived from small molecule structuresof amino acids and derivatives.16

Overall the re®ned structure represents a sub-stantial improvement as judged by geometry stat-istics and crystallographic R-factors. In Figure 1 theexcellent ®t of the re®ned model within the2Fo ÿ Fc map shows a clear improvement withrespect to the ®t of the initial model in the rawdensity (See Figure 2 in Nogales et al.4). Mostimportantly, re®nement has corrected numerous``bad'' bond angles and several signi®cant stericclashes that were present in the original, unre®nedmodel. Essentially all of the structure now fallswithin the `àllowed'' regions of a Ramanchandranplot, except for loop regions that are poorlyde®ned (residue statistics obtained with PRO-CHECK 17). While the Ramachandran plot is stillnot as good as those for typical high-resolution

Figure 1. Stereo view of part ofthe 2Fo ÿ Fc map (contoured at 1s) and the re®ned structure ofhelix H11. The Figure was gener-ated with O.45


crystal structures, it compares well with those fromother X-ray structures at comparable resolution.18

Discussion

Refinement

The crystallographic re®nement and manualrebuilding resulted in R-factors that are compar-able to those for X-ray protein structures of thissize and at this low resolution. The R-factor of 23.2and Rfree of 29.7 are the lowest yet reported for aprotein structure determined by electron crystallo-graphy (previous values include an R-factor of 28for bacteriorhodopsin (bR),13 33.0 and 37.9 (R-fac-tor and free R-factor) for the green plant light har-vesting complex (LHC-II) 19 and 39.9 and 41.7 foraquaporin (AQP-1:20 for this protein the data setused for the re®nement included very low (96 AÊ )resolution data). Electron crystallographic ampli-tudes have a much lower signal to noise ratio thantypical X-ray data, as judged by Friedel and mer-ging R-factors (Table 1). The large electron crystal-lography R-factors have thus been attributed to thelower accuracy in determining amplitudes. Majorcontributors to the low R-factors in this work arecertainly anisotropic temperature factor correctionand bulk solvent correction. Two additional factorsare near local symmetry introduced by the verysimilar structure of a and b-tubulin, and the highsolvent content introduced by sampling along c in90 AÊ intervals. The solvent content is about 70 %.Both factors introduce arti®cial coupling betweenthe amplitudes which keeps the Rfree closer to theR-factor than in a unit cell only ®lled with randomatoms. On the other hand, chemical bonding effectsare known to increase R-factors at low resolutionin electron crystallography, and it has been pro-posed that a signi®cant part of the high reportedR-factors is attributable to such effects.21 Attempts

Table 1. Electron crystallographic data and re®nement

A. Two-dimensional crystalsLayer group P121

Unit cell (refined) (AÊ ) a � 81.2, b � 93.5Thickness (AÊ ) c � 90 (assumed)

B. Electron diffractionNumber of diffraction patterns 208Resolution (AÊ ) 3.5 (in plane)

4.0 (out of plane)RFriedel (%) 19Rmerge (%) 25I/sI 5.4 (2.3 from 3.7 to 3.5 AÊ )Fourier space sampled (%) 84.7

C. ImagesImages used 149Average phase residual 35� (46 � from 4 to 3.7 AÊ )Fourier space sampled (%) 73

D. Crystallographic refinementR-factor (20 AÊ to 3.5 AÊ ) (%) 23.2Free R-factor (20 AÊ to 3.5 AÊ ) (%) 29.7

to take bonding effects into account13,22 havesuggested that the R-factors could be improved atlow resolution and that useful information aboutcharge states could be obtained by including theeffects in computation of structure factors from anatomic model. No attempt has been made here toaccount for bonding effects as aside from anincreased R-factor at low resolution, they shouldnot signi®cantly affect construction of the modelwithin the density map. However, the largeincrease in scattering amplitude at low scatteringangles for positively charged species probably con-tributes to our ability to identify the magnesiumand zinc ions.

During re®nement, the R-factor decreased from42.0 to 23.2. A similar measure, Rphase, can be cal-culated as a measure of the difference between cal-culated and experimental phases, and has beenfound to be a relevant parameter for electron crys-tallography. In the course of re®ning the bacterior-hodopsin structure Rphase decreased from 64 � to58 �.13 In re®ning the tubulin structure Rphase actu-ally increased from an initial value of 50.3 � to a®nal value of 54.0 �, probably indicating that theoriginal model was ®tted accurately into the exper-imental density, but that the density must havebeen biased by small errors in the experimentalphases.

Electron crystallography is limited in the abilityto obtain a complete set of structure factors. Themaximum tilt angle of 60-70 � for the specimenholder produces a ``hollow cone'' within whichdata is inaccessible. With a tilt range up to 60 �,though, only about 13 % of the data is missing.There is thus only a small anisotropy in the pointspread function, and with suf®ciently high resol-ution there is no ambiguity in interpretation of thedensity map.23 However, a more serious problemarises in practice when specimens are not perfectly¯at, causing a loss of signal perpendicular to thetilt axis with tilted specimens. Use of anisotropictemperature factors has reduced the impact of thisproblem to some extent, but this has, in fact, beenthe main resolution limit for both phases andamplitudes in the direction perpendicular to thecrystal plane.

Regions of major changes

The re®ned structure has the same overall foldas that obtained originally (Figure 2(a)), a tributeto the quality of the experimental phases. There®nement has allowed a much better de®nition ofside-chain positions, particularly in polar residueson the surface of the molecules. The RMS deviationbetween the original and re®ned Ca atoms is 2.4 AÊ

when including the long loop near the N terminusof b-tubulin, and 1.6 AÊ if this retraced loop isignored (Figure 2(b) shows the original and theretraced loop between H1 and S2 in b-tubulin).

There are small changes in the delimitation ofsecondary structure, which are not identical for thea and b subunit (Figure 3(a)), and generally corre-

Figure 2. (a) Stereo view of the Ca carbon traces for the original model (1TUB, magenta) and the re®ned model(1JFF, yellow). The stretch in the lattice parameters for the re®ned model is clearly apparent in this superposition. (b)Stereo view of the retraced loop between H1 and S2 in b-tubulin. The Figure was generated with O.45


spond to the appearance of new small helices (H10,H20, H200, H30, H90 and H110). It is not clearwhether we can interpret the small differencesbetween a and b-tubulin as signi®cant, or if theyare just due to inaccuracy given the limited resol-ution. It is however interesting to notice that maindifferences are localized in loop T2 (H20 and H200),the structural equivalent of the Switch I region inclassical GTPases (Figure 3(b)). Furthermore, helixH30 is a p-helix in b-tubulin in the present re®nedmodel, and thus `ùnwound'' with respect to thesame helix in a-tubulin. This region in a and b-

tubulins corresponds to the Switch II region inclassical GTPases. Thus it is tempting to speculatethat these small differences re¯ect the differencesin nucleotide state between the two tubulin sub-units in the model.

The major changes in the structure of the ab-tubulin after re®nement include three one residueprotein sequence frame shifts for both subunitsand the retracing of more weakly de®ned loops.The three protein sequence frame shifts of one resi-due were detected in identical positions of a andb-tubulin, involving the following residues: 81-97

Figure 3. Comparison of the re®ned structures of a and b-tubulin. (a) Secondary structure for a and b-tubulin inthe re®ned model as de®ned by dssp.46 Conserved residues are boxed in blue. New a-helical segments have beenlabeled with 0 and 00 and the number of the preceding helix. Boxed residues make direct contact with the nucleotideor the magnesium ion. (b) Stereo view of the Ca atoms for the re®ned structures of a (yellow) and b-tubulin (purple).The Figure includes nucleotides and taxol bound to b-tubulin. The Figure was generated with O.45


(83-97 in b-tubulin), which include strand B3; 175-197, which comprises loop T5 and helix H5; and400-405 (now identi®ed as a-helix H110), betweenthe C-terminal H11 and H12 helices.

As for the initial model, the re®ned structurelacks the C-terminal tails of both a and b-tubulin.These highly acidic segments are the main site ofvariability between isotypes and also the main sitefor posttranslational modi®cations.24 Although themixtures of several different isotypes and posttran-slational modi®cations in bovine brain tubulin (thetubulin source used for the crystallographic stu-

dies) may have contributed to the lack of densityin the map, it is generally believed that theseregions are extended and disordered in micro-tubules,25 and most likely also in zinc-inducedsheets.26

The poorest density area for both a and b-tubu-lin is located in loop H1-B2 near the N terminus. Inthe case of a-tubulin the re®nement did not resultin an improvement of the density, but rather a dis-appearance of part of it. Consequently residues 35-60 are not included in the model of the a-tubulin.Some improvement was, however, visible for the

Figure 4. Stacking of aromatic residues in the N-term-inal b-sheet of a-tubulin. The Figure was generated withO.45


b-subunit and this part of the structure is nowrebuilt. The path of the polypeptide chain is dis-tinctively different from that previously presentedin the unre®ned model and includes a smalla-helix (H10). Certain residues still have very pooror almost disconnected density, including His37,Gly38, Ala56, Ala57 and Gly58. The density is alsovery poor for residues 80-84 (H2-S3 loop) in both aand b-tubulin models.

Several of the other loop regions have beenimproved substantially. The M-loop, involved inlateral contacts between the proto®laments both inzinc-induced sheets and microtubules, has beenretraced. In a-tubulin the B9-B10 loop (residues363-368), containing an eight residue insertion withrespect to b-tubulin, has also been retraced, butdensities remain poor for residues Asp367, Leu368and Lys 370. Although all but one of the loops inthe tubulin dimer could be traced, the limited res-olution and the anisotropy of the data haveresulted in poor Ramachandran plots in some ofthese loops.

Two important additions to the original modelare the inclusion of one magnesium ion at thenucleotide N-site and one zinc ion at the lateralcontact between a-subunits. While the existence ofthe N-site magnesium was expected from numer-ous experimental results,.27-29 the accuracy of theposition of this atom is limited due to the anisotro-py of the density map. In the a-subunit an extradensity adjacent to the M-loop has been tentativelyassigned to a zinc atom that contributes signi®-cantly to the contact and to the stabilization of theM-loop. The identi®cation and positioning of thezinc ion is slightly more speculative. However,both the size and the residues surrounding thedensity attributed to Zn2� agree well with thisassignment.

Sequence conservation andtubulin polymerization

The high conservation of the ab-tubulinsequences across species has been interpreted as aconsequence of the restrictions imposed by tubulinself-assembly. Mutation of a residue in a surfaceinvolved in tubulin-tubulin contacts could dramati-cally affect polymerization unless a number ofcoordinated mutations were to occur simul-taneously across the interface to compensate forthe deleterious effect. It is most interesting howthis hypothesis is rati®ed by the present tubulinmodel. The larger stretches of absolutely conservedresidues (taken from Burns & Surridge30) cluster ina well de®ned area across the longitudinal inter-face between tubulin subunits. This region corre-sponds to the T3 loop and the loop and small helixH110 between H11 and H12, on one side, and helixH8 on the other. This region is particularly wellde®ned in the electron density map and thus theaccuracy of this part of the a and b-tubulin chainsis especially good. In contrast, residues involved inlateral contacts cluster in regions of divergence

between species and marked differences between aand b-tubulins. This suggests that both variabilityin proto®lament numbers and, most generally,marked differences in dynamic instability, seem tobe obtained by variations in residues involved incontacts between proto®laments.

Monomer stability

Tubulin is particularly rich in aromatic residues.A series of aromatic residues contribute to the stab-ility and robustness of the nucleotide bindingdomain as they align and stack against each otherin the beta sheet (Figure 4).

Of particular interest is the region of contactbetween the N-terminal and the second domain inb-tubulin. A relative rotation of these domains hasbeen proposed to be linked to microtubule depoly-merization following nucleotide hydrolysis.31

Essential to this interface are residues 198 to 216including strand S6 and helix H6. This section ofthe N-terminal domain interacts with the core helixH7, and with several regions in the intermediatedomain, specially strand S7 and the long loop fol-lowing helix H9. Of particular interest is b:Tyr202.The side-chain hydrogen bonds to Asn167 andGlu200 in an otherwise very hydrophobic region.In a-tubulin the tyrosine is substituted by aphenylalanine that makes non-polar interactionswith Leu167 and Cys200. In fungi b-tubulin alsohas a phenylalanine at position 202, and accord-ingly residue 167 is an alanine while there is noconsensus for position 200. In Saccharomyces cerevi-siae mutation of Glu200 to alanine results in beno-myl resistance,11 suggesting that such changeresults in impaired drug binding or, more likely, ina gain in microtubule stability. Interestingly, Ant-arctic ®sh b-tubulin have a phenylalanine in pos-ition 202, in spite of the conservation of Asn167and Glu200.32 Microtubules from these species arecold resistant and have reduced dynamics, furthersuggesting the importance of this region in thestability of microtubules via its role at the domainboundary in b-tubulin.


Nucleotide binding

The conformation of the nucleotide and bindingpocket in both a and b-tubulin have improvedwith the re®nement (Figure 5). Residues directly incontact with the nucleotide have been boxed inFigure 3. The speci®city of tubulin for GTP isobtained by the hydrogen bonding of the 2-exocyc-lic amino group in GTP to the hydroxyl groups ofAsn206 and Asn228, and by hydrogen bonding ofthe 6-oxo group to the amino group of Asn206.These interactions are in good agreement with pre-dictions based on af®nity binding experiments.33

Otherwise, the base sits in a rather hydrophobicpocket de®ned by Ile16, Val131, Leu227 andVal231 on one side and Tyr224 on the other. Theribose group interacts with main chain and side-chain groups in the T5 loop, while interaction withthe phosphates is dominated by hydrogen bondswith the main chain amines in T1 (the equivalentof the P-loop in G proteins) and T4 (the glycine-rich, tubulin signature motif).

Generally residues involved directly in nucleo-tide binding and hydrolysis are highly conservedbetween a and b-subunits, with some interestingexceptions. Residue a:Ala12 is b:Cys12; residuea:Val74 in H2, involved in phosphate binding, issubstituted by b:Thr74; residue a:Phe141 in theglycine-rich T4 loop, is substituted by a b:Leu141.The sugar-binding T5 loop is very different for aand b-subunits, with a:Ile171, Tyr172, Pro173,Ala174, Thr179 and Ala180, being substituted inb-tubulin by Val171, Val172, Pro173, Ser174,Asp179 and Thr180. The most signi®cant changebetween the N-site and E-site is at position 254 inH8, which is a Glu in a-tubulin and a Lys in b-tubulin. Most of these residues, particularly thosein T5, are involved in longitudinal contactsbetween subunits and these difference are probablyimportant for the relative strength and the reversi-bility of the monomer-monomer and dimer-dimer

Figure 5. Nucleotide-binding site in a-tubulin with residumagnesium ion. The Figure was generated with Insight II (Bi

contacts, as well as the difference in nucleotidehydrolysis in the two regions. While Asp254 ina-tubulin is in an ideal position to be involved inthe hydrolysis of the E-site nucleotide, Lys254 inb-tubulin is likely to strengthen the monomer-monomer contacts through its interaction with thephosphate groups of the N-site nucleotide. Inaddition to this very signi®cant difference, othersequence differences between a and b-tubulin maycontribute to the added stability of the intradimerversus the interdimer contacts. In particular thereare a number of hydrophobic residues at the intra-dimer interface that are hydrophilic for the dimer-dimer contact: b:Ile347 (versus a:Cys347), b:Val257(versus a:Thr257), or a:100Ala (versus b:100Gly).These hydrophobic residues are likely to contributeto the instability of the tubulin monomers.

The overall conservation of residues involved innucleotide binding and hydrolysis extends to othertubulin isoforms as well as the bacterial homol-ogue FtsZ. Interestingly, there are exceptionswhere some of those motifs drastically divergefrom the consensus. A dramatic sequence deviationconcerning a highly conserved tubulin motif occursfor d-tubulins in loop T7, emphasizing the diver-gence of this tubulin with respect to the rest. Theconsensus sequence GxxNxD is changed in d-tubu-lins to YxxN-P. The GxxNxD motif is a marker oflongitudinal interactions between tubulins andFtsZs. Another striking change occurs in g-tubulinwith a Gly-Gly insertion in the T3 loop preceding ahighly conserved sequence within the tubulinfamily (GNNWAxG). Finally loop T5, involvedboth in nucleotide-ribose binding and longitudinalcontacts along proto®laments, is a region of signi®-cant variability in sequence and length amongtubulins. This variability is likely to affect the abil-ity of these tubulin isoforms to make longitudinalcontacts with other tubulin molecules.34

In the re®ned structure a magnesium ion hasbeen built into extra density at the N-site. No

es involved in direct interactions with the GTP and theosym Inc.).


equivalent density could be seen at the E-sitewhere the nucleotide has clearly been hydrolyzed.It has been known for a while that GTP-tubulinhas two tightly bound Mg2� (at the N- andE-sites), whereas GDP-tubulin has a single highaf®nity Mg2�.27 The magnesium ion at the N-sitecontrols the stability and structure of the ab-tubulin.29 At the E-site Mg2� is known to be tightlylinked to the binding of GTP.35 Upon polymeriz-ation there is a reduction in the Mg2� content oftubulin, indicating its loss during GTP hydro-lysis.36 The present position of the N-site mag-nesium agrees well with the position of amagnesium atom in the crystal structure of FtsZbound to a non-hydrolizable GTP analogue (S.C.Cordell and J.L., unpublished results). In tubulinthis magnesium ion is bound by salt bridges with

Figure 6. (a) 2Fo ÿ Fc density for taxol within the b-tubulingenerated with O.45 (b) Stereo view of the taxol site includiecule. The Figure was generated with Insight II (Biosym Inc.)

two highly conserved residues, Asp69 and Glu71in the T2 loop.

Taxol binding

The density corresponding to taxol in the2Fo ÿ Fc map is much better de®ned than for theraw density for the taxane ring, the core of thetaxol molecule (Figure 6(a)). Interestingly, the den-sities for both the 2-phenyl side-chain and the N0phenyl group remain low through the re®nement,suggesting certain mobility of these groups. Whilevariation in the N0 group seem to have little effecton the binding and activity of taxol derivatives, the2-phenyl side-chain is absolutely required forfunction.37 It is thus surprising that this part of themolecule is not more ordered. The re®ned taxolstructure is in a conformation very similar to that

structure shown as Ca trace for clarity. The Figure wasng residues that make direct contact with the taxol mol-.


determined independently by energy-basedre®nement,12 except for torsional rotations of theside-chain phenyl rings. In addition, the M-loop ofboth tubulin subunits has been redrawn, resultingin a slightly different binding pocket.

A considerable number of residues, mostly inthe second domain of b-tubulin, are involved indirect contact with the anticancer drug taxol(Figure 6(b)). In helix H1, Val23 makes hydro-phobic contact with both the N0 and 30 phenylrings, while Asp26 is in hydrogen bonding dis-tance from the nitrogen group in the side-chain. Inthe H6-H7 loop, Leu217 and Leu219 make hydro-phobic contact with the 2-phenyl ring, which iscompleted by His229 and Leu230 in the core helix(H7). In the same helix Ala233 and Ser236 contactthe 30-phenyl group. The hydrophobic environmentof the 30phenyl group is completed by Phe272 at

Figure 7. Zinc binding. (a) A putative zinc ion (green) hasubunits, which interacts with the M-loop of the subunit onright. The arrows point to the plus end of the proto®lamentsdues involved in Zn2� binding: His283 and Glu284 on the MFigure was generated with Insight II (Biosym Inc.).

the end of the B7 strand. Essential for the bindingof the taxane ring is the M-loop, in particular resi-dues Pro274, Leu275, Thr276 (which contacts theessential oxetane ring), Ser277 and Arg278. Thepocket is completed by residues in the B9-B10 loop(Pro360, Arg369, Gly370 and Leu371).

Lateral contacts

Lateral contacts between proto®laments in thezinc sheets entail extensive interactions betweenhomologous subunits(a-a, b-b). The tightest part ofthe interface involves the interaction of the M-loopin one subunit with helices H12 and H5 in theadjacent one. H12 has been identi®ed as a majorsite for interaction of tubulin with motor proteins.38

The involvement of H12 in lateral interactions inthe zinc sheets explains the poor binding of kine-

s been modeled at the lateral contacts between a-tubulinthe left and helices H12 and H5 of the subunit on theas de®ned for microtubules. (b) Stereo view of the resi-

-loop, Glu 417 in helix H12 and His192 in helix H5. The


sin-like motors to these polymers, and their desta-bilizing effect at high concentrations.39,40

The lateral contact is more clear between a-sub-units as their M-loop is more ordered and thus bet-ter de®ned in the model. In this case, a zinc ionhas been modeled into the structure, sitting by resi-dues a:His283, a:Glu284 and a:Gln285 of the M-loop (Figure 7). Residues interacting with the zincion at the other side of the interface includea:His192 in helix H5 and a:Glu417 in helix H12,with other acidic residues in H12 also near the site(a:Glu420, a:Glu423 and a:Asp424). His283 andGlu284 are totally conserved residues in a-tubulins(which are substituted by weakly conserved Tyrand Arg in b-tubulins). His192, and all the men-tioned residues in H12 are also totally conserved ina-tubulins. The location of zinc and its active,direct participation in lateral interactions stronglysuggests that this zinc is as least partially respon-sible for the generation of the alternative, antiparal-lel proto®lament-proto®lament interaction in zinc-induced polymers. Although this is the only zincion we can identify, other zinc ions are probablyrequired for the formation of the sheets, perhaps inlower af®nity sites.

The involvement of histidines in the binding ofthe zinc ion agrees with the low pH (�5.8)required for the formation of the sheets. Interest-ingly, higher pH of 6.0-6.4 results in the formationof macrotubes39 by bending of the proto®lamentsaccompanied by a longitudinal shift of adjacentproto®laments.41 This suggests that one or both ofthe histidines involved in zinc binding has a pKa ofapproximately 6, and the differences between thetwo zinc-induced polymers has to do with differ-ences in the protonation of one or both of these his-tidine residues.

Lateral contacts also include the interaction ofthe last residues in helix H6, helix H9 and the fol-lowing loop, and H10, with residues in helices H4and H3 in the adjacent subunit. This contact ismostly polar and ionic, involving the interaction ofresidues a: Arg214, Arg215 and Asn216 in H6 withresidues a:Arg156, Val159, Asp160 and Lys163 inH4; and residues a:Asn293 and Glu297 (in H9 andthe following loop) with Arg156. The network ofinteractions extends, through Asn293, to Lys112and Asp116 in H3, the latter also within interactiondistance from Lys338 at the end of helix H10.These residues are not absolutely conserved acrossspecies.

Conclusions

In the re®ned model of ab-tubulin two loopsessential for proto®lament interactions in themicrotubule, the M-loop and the loop between H1and S2, have been retraced. A magnesium ion isnow modeled within the N-site of a-tubulin. Azinc ion, essential for the formation of the zinc-sheets used for the crystallographic study, has nowbeen localized at lateral contacts between a-tubulin

subunits. Most importantly, the re®nement of theab-tubulin structure has resulted in a muchimproved geometry and the correction of threesmall frame shifts. Therefore, this new structure isa signi®cant improvement from the original model,and most relevant for studies involved with themodeling of drug-binding sites on tubulin andwith structural changes in tubulin that relate todynamic instability.

Materials and Methods

Phases for the initial 3.7 AÊ model were obtained from149 images, 86 of which were taken at 55-60 � tilt.4 Thephase residual for this data set was 30 � or better up to5 AÊ resolution, 37 � between 4 and 5 AÊ , and 46 � for thehighest resolution shell. Initial amplitudes were obtainedfrom 94 electron diffraction patterns, which had an over-all Friedel R-factor of 19 % and an overall Rmerge of 25 %.

The initial PDB entry for the tubulin dimer (1TUB)contained a molecule of taxotere, as obtained by X-raycrystallography, docked as a rigid body into the electrondensity corresponding to taxol. Before we initiated there®nement procedures of the tubulin dimer, we pro-duced a more accurate model of taxol as described.12

Brie¯y, 26 conformers of taxol and taxol derivativesobtained from various structural studies, including X-raycrystal structures, NMR nuclear Overhauser enhance-ment data, as well as a large number of computer-generated conformers, were ®tted into the electron crys-tallographic density associated with the ligand in b-tubu-lin. One of the best solutions was optimized in thebinding site using restricted low temperature dynamicsand force ®eld optimization, keeping the evolving modelfully consistent with the experimental density of thetubulin-taxol complex. The uniqueness and reproducibil-ity of the ®nal model were independently tested byremoving taxol from the protein, conformationally alter-ing it and ¯exibly redocking it into the binding pocket.Only two of the generated structures were encased bythe protein, and the lowest energy form was identical inshape and location to that previously obtained. Thisenergy-minimized taxol was used as a starting point forthe crystallographic re®nement.

An experimental ®gure-of-merit (FOM) for the phaseswas calculated from the deviation of experimentalphases from the curves ®t to the measurements.42 TheseFOM values proved unsuitable for re®nement as theydid not improve the convergence of re®nement. Insteadequally weighted phase probability functions derivedfrom phase angles were used.

Because the amplitude data showed a rather highinternal temperature factor when ®tted in a Wilson plot(approx 65 AÊ 2), the amplitudes were sharpened by atemperature factor of ÿ25 AÊ 2. Five percent of re¯ectionswere selected randomly and were used throughout there®nement for the calculation of Rfree. Phases were con-verted into unimodal Hendrickson-Lattmann coef®cientsand blurred by a scale factor of 0.2 and a temperaturefactor of 50.0. These numbers were found empirically bya two-dimensional optimization of Rfree after maximum-likelihood positional re®nement. Atomic scattering fac-tors for electrons43 were used without modi®cation andwithout taking charge or chemical bonding effects intoaccount.

All re®nement procedures were carried out in CNSversion 0.9.44


Protein Data Bank accession numbers

The coordinates described here were deposited at thePDB with accession number 1JFF.

Acknowledgments

This work was supported by NIH grant GM46033(K.H.D.) and by the Of®ce of Health and EnviromentalResearch of the U.S. Department of Energy under con-tract DE.AC03.76F00098 (K.H.D. and E.N.).

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Edited by I. A. Wilson

(Received 25 June 2001; received in revised form 30 August 2001; accepted 5 September 2001)

Documents

Refined Structure of (alpha)(beta)-Tubulin at 3.5 A Resolution tubulin...improved by incorporating 114 additional diffrac-tion patterns. A few of the patterns extend to 2.5 A˚ . However