NORMAL GENOMIC

• Lappchen T et al.
• Probing FtsZ and tubulin with C8-substituted GTP analogs revealsdifferences in their nucleotide binding sites.
• Chem Biol. 2008; 15: 189-99
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• The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role inprokaryotic cell division and eukaryotic chromosome segregation,respectively. Selective inhibitors of the GTP-dependent polymerization ofFtsZ could constitute a new class of antibiotics, while several inhibitorsof tubulin are widely used in antiproliferative therapy. In this work, weset out to identify selective inhibitors of FtsZ based on the structure ofits natural ligand, GTP. We found that GTP analogs with small hydrophobicsubstituents at C8 of the nucleobase efficiently inhibit FtsZpolymerization, whereas they have an opposite effect on the polymerizationof tubulin. The inhibitory activity of the GTP analogs on FtsZpolymerization allowed us to crystallize FtsZ in complex withC8-morpholino-GTP, revealing the binding mode of a GTP derivativecontaining a nonmodified triphosphate chain.

• Huecas S, Llorca O, Boskovic J, Martin-Benito J, Valpuesta JM, Andreu JM
• Energetics and geometry of FtsZ polymers: nucleated self-assembly ofsingle protofilaments.
• Biophys J. 2008; 94: 1796-806
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• Essential cell division protein FtsZ is an assembling GTPase which directsthe cytokinetic ring formation in dividing bacterial cells. FtsZ sharesthe structural fold of eukaryotic tubulin and assembles formingtubulin-like protofilaments, but does not form microtubules. Two puzzlingproblems in FtsZ assembly are the nature of protofilament association anda possible mechanism for nucleated self-assembly of single-strandedprotofilaments above a critical FtsZ concentration. We assembledtwo-dimensional arrays of FtsZ on carbon supports, studied linear polymersof FtsZ with cryo-electron microscopy of vitrified unsupported solutions,and formulated possible polymerization models. Nucleated self-assembly ofFtsZ from Escherichia coli with GTP and magnesium produces flexiblefilaments 4-6 nm-wide, only compatible with a single protofilament. Thisagrees with previous scanning transmission electron microscopy results andis supported by recent cryo-electron tomography studies of two bacterialcells. Observations of double-stranded FtsZ filaments in negative stainmay come from protofilament accretion on the carbon support. Preferentialprotofilament cyclization does not apply to FtsZ assembly. The apparentlycooperative polymerization of a single protofilament with identicalintermonomer contacts is explained by the switching of one inactivemonomer into the active structure preceding association of the next,creating a dimer nucleus. FtsZ behaves as a cooperative linear assemblymachine.

• Demchuk ON, Nyporko AIu, Blium IaB
• [Construction of three-dimensional models of Arabidopsis thalianaFtsZ-proteins on basis of crystal structure of archaebacterial FtsZ-GDPcomplex]
• Tsitol Genet. 2006; 40: 10-20
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• Three-dimensional models of FtsZ-protein complexes with GDP fromArabidopsis thaliana L. localized in cytosol (Entrez database codeNP190843) and in chloroplasts (Entrez database code AAA82068) weredeveloped. Crystal structure of the FtsZ-GDP complex from archaeaMethanococcus jannaschii (PDB-code 1FSZ) was used as a matrix. Secondarystructures of computed models contain ten beta-strands. A chloroplast formof FtsZ-protein has ten alpha-helices and four 3(10)-helices, whereascytosolic form of protein has nine and three structures correspondentlyand neither a0-helix before nucleotide-binding domain nor C-terminal3(10)-helix in secondary domain. The T2-loop of nucleotide-binding pocketof chloroplast form of FtsZ-ptotein in position 111 contains non-chargedalanin residue instead of the charged one which is typical for cytosolicand bacterial forms of proteins. At low sequence homology of FtsZ-proteins(approximately 47%) the developed models demonstrate high coincidence withmatrix both in the structures of nucleotide-binding pocket and in thewhole molecule. The models are completely suitable for further studies ofpossible sites of binding with dinitroaniline herbicides.

• Leung AK, Lucile White E, Ross LJ, Reynolds RC, DeVito JA, Borhani DW
• Structure of Mycobacterium tuberculosis FtsZ reveals unexpected, Gprotein-like conformational switches.
• J Mol Biol. 2004; 342: 953-70
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• We report three crystal structures of the Mycobacterium tuberculosis celldivision protein FtsZ, as the citrate, GDP, and GTPgammaS complexes,determined at 1.89, 2.60, and 2.08A resolution. MtbFtsZ crystallized as atight, laterally oriented dimer distinct from the longitudinal polymerobserved for alphabeta-tubulin. Mutational data on Escherichia coli FtsZsuggest that this dimer interface is important for proper protofilamentand "Z-ring" assembly and function. An alpha-to-beta secondary structureconformational switch at the dimer interface is spatially analogous to,and has many of the hallmarks of, the Switch I conformational changesexhibited by G-proteins upon activation. The presence of a gamma-phosphatein the FtsZ active site modulates the conformation of the "tubulin" loopT3 (spatially analogous to the G-protein Switch II); T3 switching upongamma-phosphate ligation is directly coupled to the alpha-to-beta switchby steric overlap. The dual conformational switches observed here for thefirst time in an FtsZ link GTP binding and hydrolysis to FtsZ (andtubulin) lateral assembly and Z-ring contraction, and they are suggestiveof an underappreciated functional analogy between FtsZ, tubulin andG-proteins.

• Oliva MA, Cordell SC, Lowe J
• Structural insights into FtsZ protofilament formation.
• Nat Struct Mol Biol. 2004; 11: 1243-50
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• The prokaryotic tubulin homolog FtsZ polymerizes into a ring structureessential for bacterial cell division. We have used refolded FtsZ tocrystallize a tubulin-like protofilament. The N- and C-terminal domains oftwo consecutive subunits in the filament assemble to form the GTPase site,with the C-terminal domain providing water-polarizing residues. Adomain-swapped structure of FtsZ and biochemical data on purified N- andC-terminal domains show that they are independent. This leads to a modelof how FtsZ and tubulin polymerization evolved by fusing two domains. Inpolymerized tubulin, the nucleotide-binding pocket is occluded, whichleads to nucleotide exchange being the rate-limiting step and to dynamicinstability. In our FtsZ filament structure the nucleotide isexchangeable, explaining why, in this filament, nucleotide hydrolysis isthe rate-limiting step during FtsZ polymerization. Furthermore, crystalstructures of FtsZ in different nucleotide states reveal notably fewdifferences.

• Andreu JM, Oliva MA, Monasterio O
• Reversible unfolding of FtsZ cell division proteins from archaea andbacteria. Comparison with eukaryotic tubulin folding and assembly.
• J Biol Chem. 2002; 277: 43262-70
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• The stability, refolding, and assembly properties of FtsZ cell divisionproteins from Methanococcus jannaschii and Escherichia coli have beeninvestigated. Their guanidinium chloride unfolding has been studied bycircular dichroism spectroscopy. FtsZ from E. coli and tubulin releasedthe bound guanine nucleotide, coinciding with an initial unfolding stageat low denaturant concentrations, followed by unfolding of the apoprotein.FtsZ from M. jannaschii released its nucleotide without any detectablesecondary structural change. It unfolded in an apparently two-statetransition at larger denaturant concentrations. Isolated FtsZ polypeptidechains were capable of spontaneous refolding and GTP-dependent assembly.The homologous eukaryotic tubulin monomers misfold in solution, but foldwithin the cytosolic chaperonin CCT. Analysis of the extensive tubulinloop insertions in the FtsZ/tubulin common core and of the intermolecularcontacts in model microtubules and tubulin-CCT complexes shows a loopinsertion present at every element of lateral protofilament contact and atevery contact of tubulin with CCT (except at loop T7). The polymers formedby purified FtsZ have a distinct limited protofilament association incomparison with microtubules. We propose that the loop insertions oftubulin and its CCT-assisted folding coevolved with the lateralassociation interfaces responsible for extended two-dimensionalpolymerization into microtubule polymers.

• Keskin O, Durell SR, Bahar I, Jernigan RL, Covell DG
• Relating molecular flexibility to function: a case study of tubulin.
• Biophys J. 2002; 83: 663-80
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• Microtubules (MT), along with a variety of associated motor proteins, areinvolved in a range of cellular functions including vesicle movement,chromosome segregation, and cell motility. MTs are assemblies ofheterodimeric proteins, alpha beta-tubulins, the structure of which hasbeen determined by electron crystallography of zinc-induced,pacilitaxel-stabilized tubulin sheets. These data provide a basis forexamining relationships between structural features and protein function.Here, we study the fluctuation dynamics of the tubulin dimer with the aimof elucidating its functional motions relevant to substrate binding,polymerization/depolymerization and MT assembly. A coarse-grained model,harmonically constrained according to the crystal structure, is used toexplore the global dynamics of the dimer. Our results identify six regionsof collective motion, comprised of structurally close but discontinuoussequence fragments, observed only in the dimeric form, dimerization beinga prerequisite for domain identification. Boundaries between regions ofcollective motions appear to act as linkages, found primarily withinsecondary-structure elements that lack sequence conservation, but arelocated at minima in the fluctuation curve, at positions of hydrophobicresidues. Residue fluctuations within these domains identify the mostmobile regions as loops involved in recognition of the adjacent regions.The least mobile regions are associated with nucleotide binding siteswhere lethal mutations occur. The functional coupling of motions betweenand within regions identifies three global motions: torsional and wobblingmovements, en bloc, between the alpha- and beta-tubulin monomers, andstretching longitudinally. Further analysis finds the antitumor drugpacilitaxel (TaxotereR) to reduce flexibility in the M loop of thebeta-tubulin monomer; an effect that may contribute to tightening lateralinteractions between protofilaments assembled into MTs. Our analysisprovides insights into relationships between intramolecular tubulinmovements of MT organization and function.

• Scheffers DJ, de Wit JG, den Blaauwen T, Driessen AJ
• GTP hydrolysis of cell division protein FtsZ: evidence that the activesite is formed by the association of monomers.
• Biochemistry. 2002; 41: 521-9
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• The essential prokaryotic cell division protein FtsZ is a tubulinhomologue that forms a ring at the division site. FtsZ forms polymers in aGTP-dependent manner. Recent biochemical evidence has shown that FtsZforms multimeric structures in vitro and in vivo and functions as aself-activating GTPase. Structural analysis of FtsZ points to an importantrole for the highly conserved tubulin-like loop 7 (T7-loop) in theself-activation of GTP hydrolysis. The T7-loop was postulated to form theactive site together with the nucleotide-binding site on an adjacent FtsZmonomer. To characterize the role of the T7-loop of Escherichia coli FtsZ,we have mutagenized residues M206, N207, D209, D212, and R214. All themutant proteins, except the R214 mutant, are severely affected inpolymerization and GTP hydrolysis. Charged residues D209 and D212 cannotbe substituted with a glutamate residue. All mutants interact withwild-type FtsZ in vitro, indicating that the T7-loop mutations do notabolish FtsZ self-association. Strikingly, in mixtures of wild-type andmutant proteins, most mutants are capable of inhibiting wild-type GTPhydrolysis. We conclude that the T7-loop is part of the active site forGTP hydrolysis, formed by the association of two FtsZ monomers.

• Diaz JF, Kralicek A, Mingorance J, Palacios JM, Vicente M, Andreu JM
• Activation of cell division protein FtsZ. Control of switch loop T3conformation by the nucleotide gamma-phosphate.
• J Biol Chem. 2001; 276: 17307-15
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• The effect of bound nucleotide on the conformation of cell divisionprotein FtsZ from Methanococcus jannaschii has been investigated usingmolecular dynamics and site-directed mutagenesis. The molecular dynamicsindicate that the gamma-phosphate of GTP induces a conformationalperturbation in loop T3 (Gly88-Gly99 segment), in a position structurallyequivalent to switch II of Ha-ras-p21. In the simulated GTP-bound state,loop T3 is pulled by the gamma-phosphate into a more compact conformationthan with GDP, related to that observed in the homologous proteins alpha-and beta-tubulin. The existence of a nucleotide-induced structural changein loop T3 has been confirmed by mutating Thr92 into Trp (T92W-W319YFtsZ). This tryptophan (12 A away from gamma-phosphate) shows largedifferences in fluorescence emission, depending on which nucleotide isbound to FtsZ monomers. Loop T3 is located at a side of the contactinterface between two FtsZ monomers in the current model of FtsZ filament.Such a structural change may bend the GDP filament upon hydrolysis bypushing against helix H8 of next monomer, thus, generating force on themembrane during cell division. A related curvature mechanism may operatein tubulin activation.

• Lowe J, Li H, Downing KH, Nogales E
• Refined structure of alpha beta-tubulin at 3.5 A resolution.
• J Mol Biol. 2001; 313: 1045-57
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• We present a refined model of the alpha beta-tubulin dimer to 3.5 Aresolution. An improved experimental density for the zinc-induced tubulinsheets was obtained by adding 114 electron diffraction patterns at 40-60degrees tilt and increasing the completeness of structure factoramplitudes to 84.7 %. The refined structure was obtained usingmaximum-likelihood including phase information from experimental images,and simulated annealing Cartesian refinement to an R-factor of 23.2 andfree R-factor of 29.7. The current model includes residues alpha:2-34,alpha:61-439, beta:2-437, one molecule of GTP, one of GDP, and one oftaxol, as well as one magnesium ion at the non-exchangeable nucleotidesite, and one putative zinc ion near the M-loop in the alpha-tubulinsubunit. The acidic C-terminal tails could not be traced accurately,neither could the N-terminal loop including residues 35-60 in thealpha-subunit. There are no major changes in the overall fold of tubulinwith respect to the previous structure, testifying to the quality of theinitial 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 defined. Three shortprotein sequence frame shifts were detected with respect to thenon-refined structure. In light of the new model we discuss details of thetubulin structure such as nucleotide and taxol binding sites, lateralcontacts in zinc-sheets, and the significance of the location of highlyconserved residues.

• Mosyak L et al.
• The bacterial cell-division protein ZipA and its interaction with an FtsZfragment revealed by X-ray crystallography.
• EMBO J. 2000; 19: 3179-91
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• In Escherichia coli, FtsZ, a homologue of eukaryotic tubulins, and ZipA, amembrane-anchored protein that binds to FtsZ, are two essential componentsof the septal ring structure that mediates cell division. Recent dataindicate that ZipA is involved in the assembly of the ring by linking FtsZto the cytoplasmic membrane and that the ZipA-FtsZ interaction is mediatedby their C-terminal domains. We present the X-ray crystal structures ofthe C-terminal FtsZ-binding domain of ZipA and a complex between thisdomain and a C-terminal fragment of FtsZ. The ZipA domain is asix-stranded beta-sheet packed against three alpha-helices and containsthe split beta-alpha-beta motif found in many RNA-binding proteins. Theuncovered side of the sheet incorporates a shallow hydrophobic cavityexposed to solvent. In the complex, the 17-residue FtsZ fragment occupiesthis entire cavity of ZipA and binds as an extended beta-strand followedby alpha-helix. An alanine-scanning mutagenesis analysis of the FtsZfragment was also performed, which shows that only a small cluster of theburied FtsZ side chains is critical in binding to ZipA.

• Hale CA, Rhee AC, de Boer PA
• ZipA-induced bundling of FtsZ polymers mediated by an interaction betweenC-terminal domains.
• J Bacteriol. 2000; 182: 5153-66
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• FtsZ and ZipA are essential components of the septal ring apparatus, whichmediates cell division in Escherichia coli. FtsZ is a cytoplasmictubulin-like GTPase that forms protofilament-like homopolymers in vitro.In the cell, the protein assembles into a ring structure at theprospective division site early in the division cycle, and this marks thefirst recognized event in the assembly of the septal ring. ZipA is aninner membrane protein which is recruited to the nascent septal ring at avery early stage through a direct interaction with FtsZ. Using affinityblotting and protein localization techniques, we have determined whichdomain on each protein is both sufficient and required for the interactionbetween the two proteins in vitro as well as in vivo. The results showthat ZipA binds to residues confined to the 20 C-terminal amino acids ofFtsZ. The FtsZ binding (FZB) domain of ZipA is significantly larger andencompasses the C-terminal 143 residues of ZipA. Significantly, we findthat the FZB domain of ZipA is also required and sufficient to inducedramatic bundling of FtsZ protofilaments in vitro. Consistent with thenotion that the ability to bind and bundle FtsZ polymers is essential tothe function of ZipA, we find that ZipA derivatives lacking an intact FZBdomain fail to support cell division in cells depleted for the nativeprotein. Interestingly, ZipA derivatives which do contain an intact FZBdomain but which lack the N-terminal membrane anchor or in which thisanchor is replaced with the heterologous anchor of the DjlA protein alsofail to rescue ZipA(-) cells. Thus, in addition to the C-terminal FZBdomain, the N-terminal domain of ZipA is required for ZipA function.Furthermore, the essential properties of the N domain may be more specificthan merely acting as a membrane anchor.

• Han Y, Sablin EP, Nogales E, Fletterick RJ, Downing KH
• Visualizing a new binding site of ncd-motor domain on tubulin.
• J Struct Biol. 1999; 128: 26-33
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• Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.

• Lowe J, Amos LA
• Tubulin-like protofilaments in Ca2+-induced FtsZ sheets.
• EMBO J. 1999; 18: 2364-71
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• The 40 kDa protein FtsZ is a major septum-forming component of bacterialcell division. Early during cytokinesis at midcell, FtsZ forms acytokinetic ring that constricts as septation progresses. FtsZ has a highpropensity to polymerize in vitro into various structures, includingsheets and filaments, in a GTP-dependent manner. Together with limitedsequence homology, the occurrence of the tubulin signature motif in FtsZand a similar three-dimensional structure, this leads to the conclusionthat FtsZ is the bacterial tubulin homologue. We have polymerized FtsZ1from Methanococcus jannaschii in the presence of millimolar concentrationsof Ca2+ ions to produce two-dimensional crystals of plane group P2221.Most of the protein precipitates and forms filaments approximately 23.0 nmin diameter. A three-dimensional reconstruction of tilted micrographs ofFtsZ sheets in negative stain between 0 and 60 degrees showsprotofilaments of FtsZ running along the sheet axis. Pairs of parallelFtsZ protofilaments associate in an antiparallel fashion to form atwo-dimensional sheet. The antiparallel arrangement is believed togenerate flat sheets instead of the curved filaments seen in other FtsZpolymers. Together with the subunit spacing along the protofilament axis,a fitting of the FtsZ crystal structure into the reconstruction suggests aprotofilamant structure very similar to that of tubulin protofilaments.

• Nogales E, Downing KH, Amos LA, Lowe J
• Tubulin and FtsZ form a distinct family of GTPases.
• Nat Struct Biol. 1998; 5: 451-8
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• Tubulin and FtsZ share a common fold of two domains connected by a central helix. Structure-based sequence alignment shows that common residues localize in the nucleotide-binding site and a region that interacts with the nucleotide of the next tubulin subunit in the protofilament, suggesting that tubulin and FtsZ use similar contacts to form filaments. Surfaces that would make lateral interactions between protofilaments or interact with motor proteins are, however, different. The highly conserved nucleotide-binding sites of tubulin and FtsZ clearly differ from those of EF-Tu and other GTPases, while resembling the nucleotide site of glyceraldehyde-3-phosphate dehydrogenase. Thus, tubulin and FtsZ form a distinct family of GTP-hydrolyzing proteins.

• Menendez M, Rivas G, Diaz JF, Andreu JM
• Control of the structural stability of the tubulin dimer by one highaffinity bound magnesium ion at nucleotide N-site.
• J Biol Chem. 1998; 273: 167-76
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• Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDPat the E-site in the beta-subunit (GDP-tubulin) reversibly binds one highaffinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded withGTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. Thetwo cation binding loci are identified as nucleotide sites N and E,respectively. Mg2+ at the N-site controls the stability and structure ofthe alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slowrelease of bound nucleotide and functional inactivation. Mg2+ bound to theN-site significantly increases the thermal stability of the GDP-tubulindimer (by 10 degrees C and approximately 50 kcal mol-1 of experimentalenthalpy change). However, the thermal stability of Mg2+-liganded GDP- andGTP-tubulin is the same. Mg2+ binding to the N-site is linked to thealphabeta-dimer formation. The binding of Mg2+ to the alpha-subunitcommunicates a marked enhancement of fluorescence to a colchicine analoguebound to the beta-subunit. Colchicine, in turn, thermally stabilizesMg2+-depleted tubulin. The tubulin properties described would be simplyexplained if the N-site and the colchicine site are at the alpha-betadimerization interface. It follows that the E-site would be at thebeta-end of the tubulin dimer, consistent with the known functional roleof the E nucleotide gamma-phosphate and coordinated cation controllingmicrotubule stability.

• Downing KH, Nogales E
• New insights into microtubule structure and function from the atomic modelof tubulin.
• Eur Biophys J. 1998; 27: 431-6
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• The structure of tubulin has recently been solved by electroncrystallography of zinc-induced tubulin sheets. Because tubulin wasstudied in a polymerized state, the model contains information on theinteractions between monomers that give rise to the alpha beta dimer aswell as contacts between adjacent dimers that result in the structure ofthe protofilament. The model includes the binding site of taxol, ananti-cancer agent that acts by stabilizing microtubules. The presenttubulin model gives the first structural framework for understandingmicrotubule polymerization and its regulation by nucleotides andanti-mitotic drugs at the molecular level.

• Lowe J
• Crystal structure determination of FtsZ from Methanococcus jannaschii.
• J Struct Biol. 1998; 124: 235-43
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• FtsZ is the polymer-forming protein of bacterial cell division. It is partof a ring in the middle of the dividing cell that is required forconstriction of cell membrane and cell envelope to yield two daughtercells. FtsZ is a GTPase and is the only bacterial protein showingsignificant sequence homology to the eukaryotic tubulins. FtsZ canpolymerize into tubes, sheets, and rings in vitro and is ubiquitous ineubacteria and archaea. Full-length FtsZ1 from Methanococcus jannaschiihas been over expressed in Escherichia coli, employing thehyperthermophilic properties of the protein. Crystals grown from PEG400and ethanol belong to spacegroup I213 with a = b = c = 159.1 A.Isomorphous replacement using one Hg derivative yielded a interpretableelectron density map at 4 A resolution. The structure for residues 23-356and one GDP has been refined to an Rfree of 0.28 (Rf = 0.20) at 2.8 Aresolution. FtsZ consists of two domains with a connecting core helix. TheN-terminal domain and the core helix contain all residues involved innucleotide binding and resemble the fold of dinucleotide-binding proteins.The structures of tubulin and FtsZ show striking similarity; together withthe functional similarities, this provides a strong indication that FtsZis a true homolog of tubulin.

• Vulevic B, Correia JJ
• Thermodynamic and structural analysis of microtubule assembly: the role ofGTP hydrolysis.
• Biophys J. 1997; 72: 1357-75
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• Different models have been proposed that link the tubulin heterodimernucleotide content and the role of GTP hydrolysis with microtubuleassembly and dynamics. Here we compare the thermodynamics of microtubuleassembly as a function of nucleotide content by van't Hoff analysis. Thethermodynamic parameters of tubulin assembly in 30-100 mMpiperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM MgSO4, 2 mM EGTA, pH 6.9,in the presence of a weakly hydrolyzable analog, GMPCPP, the dinucleotideanalog GMPCP plus 2 M glycerol, and GTP plus 2 M glycerol were obtainedtogether with data for taxol-GTP/GDP tubulin assembly (GMPCPP and GMPCPare the GTP and GDP nucleotide analogs where the alpha beta oxygen hasbeen replaced by a methylene, -CH2-). All of the processes studied arecharacterized by a positive enthalpy, a positive entropy, and a large,negative heat capacity change. GMPCP-induced assembly has the largestnegative heat capacity change and GMPCPP has the second largest, whereasGTP/2 M glycerol- and taxol-induced assembly have more positive values,respectively. A large, negative heat capacity is most consistent with theburial of water-accessible hydrophobic surface area, which gives rise tothe release of bound water. The heat capacity changes observed with GTP/2M glycerol-induced and with taxol-induced assembly are very similar, -790+/- 190 cal/mol/k, and correspond to the burial of 3330 +/- 820 A2 ofnonpolar surface area. This value is shown to be very similar to anestimate of the buried nonpolar surface in a reconstructed microtubulelattice. Polymerization data from GMPCP- and GMPCPP-induced assembly areconsistent with buried nonpolar surface areas that are 3 and 6 timeslarger. A linear enthalpy-entropy and enthalpy-free energy plot fortubulin polymerization reactions verifies that enthalpy-entropycompensation for this system is based upon true biochemical correlation,most likely corresponding to a dominant hydrophobic effect. Entropyanalysis suggests that assembly with GTP/2 M glycerol and with taxol isconsistent with conformational rearrangements in 3-6% of the total aminoacids in the heterodimer. In addition, taxol binding contributes to thethermodynamics of the overall process by reducing the delta H degree anddelta S degree for microtubule assembly. In the presence of GMPCPP orGMPCP, tubulin subunits associate with extensive conformationalrearrangement, corresponding to 10% and 26% of the total amino acids inthe heterodimer, respectively, which gives rise to a large loss ofconfigurational entropy. An alternative, and probably preferable,interpretation of these data is that, especially with GMPCP-tubulin,additional isomerization or protonation events are induced by the presenceof the methylene moiety and linked to microtubule assembly. Structuralanalysis shows that GTP hydrolysis is not required for sheet closure intoa microtubule cylinder, but only increases the probability of this eventoccurring. Sheet extensions and sheet polymers appear to have a similaraverage length under various conditions, suggesting that the minimumcooperative unit for closure of sheets into a microtubule cylinder isapproximately 400 nm long. Because of their low level of occurrence,sheets are not expected to significantly affect the thermodynamics ofassembly.

• Wolf SG, Nogales E, Kikkawa M, Gratzinger D, Hirokawa N, Downing KH
• Interpreting a medium-resolution model of tubulin: comparison ofzinc-sheet and microtubule structure.
• J Mol Biol. 1996; 262: 485-501
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• We previously used electron crystallography of zinc-inducedtwo-dimensional crystalline sheets of tubulin to construct amedium-resolution three dimensional (3-D) reconstruction (at 6.5 A) ofthis protein. Here we present an improved model, and extend theinterpretation to correlate it to microtubule structure. Secondarysequence predictions and projection density maps of subtilisin-cleavedtubulin provide information on the location of the C-terminal portion,which has been suggested to be involved in the binding ofmicrotubule-associated proteins. The zinc-sheet tubulin model is comparedto microtubules in two ways; comparison of electron diffraction from thezinc-sheets to electron diffraction from microtubules, and by docking thezinc-sheet protofilament 3-D model into a helical reconstruction fromice-embedded microtubules. By correlating the zinc-sheet protofilament toa reconstruction of axonemal protofilaments, we assigned polarity to theprotofilament in our model. The polarity assignment together with ourmodel for dimer boundaries and the assignment of alpha- and beta-monomersin our reconstruction, provides a microtubule model where thealpha-monomer crowns the plus- (or fast-growing) end of the microtubuleand contact is made in the centrosome with gamma-tubulin via thebeta-monomer.

• Erickson HP, Taylor DW, Taylor KA, Bramhill D
• Bacterial cell division protein FtsZ assembles into protofilament sheetsand minirings, structural homologs of tubulin polymers.
• Proc Natl Acad Sci U S A. 1996; 93: 519-23
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• The bacterial cell division protein FtsZ is a homolog of tubulin, but ithas not been determined whether FtsZ polymers are structurally related tothe microtubule lattice. In the present study, we have obtainedhigh-resolution electron micrographs of two FtsZ polymers that showremarkable similarity to tubulin polymers. The first is a two-dimensionalsheet of protofilaments with a lattice very similar to that of themicrotubule wall. The second is a miniring, consisting of a singleprotofilament in a sharply curved, planar conformation. FtsZ minirings arevery similar to tubulin rings that are formed upon disassembly ofmicrotubules but are about half the diameter. This suggests that thecurved conformation occurs at every FtsZ subunit, but in tubulin rings theconformation occurs at either beta- or alpha-tubulin subunits but notboth. We conclude that the functional polymer of FtsZ in bacterial celldivision is a long thin sheet of protofilaments. There is sufficient FtsZin Escherichia coli to form a protofilament that encircles the cell 20times. The similarity of polymers formed by FtsZ and tubulin implies thatthe protofilament sheet is an ancient cytoskeletal system, originallyfunctioning in bacterial cell division and later modified to makemicrotubules.

• de Pereda JM, Leynadier D, Evangelio JA, Chacon P, Andreu JM
• Tubulin secondary structure analysis, limited proteolysis sites, andhomology to FtsZ.
• Biochemistry. 1996; 35: 14203-15
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• The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulindimer analyzed by six different methods indicates an average content ofapproximately 33% alpha helix, 21% beta sheet, and 45% other secondarystructure. Deconvolution of Fourier transform infrared spectra indicates24% sheet, 37% (maximum) helix, and 38% (minimum) other structure.Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14gamma-tubulin sequences and 12 sequences of the bacterial cell divisionprotein FtsZ have been employed to predict their secondary structures withthe multiple-sequence method PHD [Rost, B., & Sander, C. (1993a) J. Mol.Biol. 232, 584-599]. The predicted secondary structures average of 33%alpha helix, 24% beta sheet, and 43% loop for the alpha beta dimer. Thepredictions have been compared with sites of limited proteolysis by 12proteases at the surfaces of the heterodimer and taxol-inducedmicrotubules [de Pereda, J. M., & Andreu, J. M. (1996) Biochemistry 35,14184-14202]. From 24 experimentally determined nicking sites, 18 are atpredicted loops or at the extremes of secondary structure elements.Proteolysis zone A (including acetylable Lys40 and probably Lys60 inalpha-tubulin and Gly93 in beta-tubulin) and proteolysis zone B (extendingbetween residues 167 and 183 in both chains) are accessible inmicrotubules. Proteolysis zone C, between residues 278 and 295, becomespartially occluded in microtubules. The alpha-tubulin nicking siteArg339-Ser340 is at a loop following a predicted alpha helix inproteolysis zone D. This site is protected in taxol microtubules; however,a new tryptic site appears which is probably located at the N-terminal endof the same helix. Zone D also contains beta-tubulin Cys354, which isaccessible in microtubules. Proteolysis zone E includes the C-terminalhypervariable loops (10-20 residues) of each tubulin chain. These followthe two larger predicted helical zones (residues 372-395 and 405-432 inbeta-tubulin), which also are the longer conserved part of the alpha- andbeta-tubulin sequences. Through combination of this with other biochemicalinformation, a set of surface and distance constraints is proposed for thefolding of beta-tubulin. The FtsZ sequences are only 10-18% identical tothe tubulin sequences. However, the predicted secondary structures showtwo clearly similar (85-87 and 51-78%) regions, at tubulin positions95-175 and 305-350, corresponding to FtsZ 65-135 and 255-300,respectively. The first region is flanked by tubulin proteolysis zones Aand B. It consists of a predictedloop1-helix-loop2-sheet-loop3-helix-loop4-sheet fold, which contains themotif (KR)GXXXXG (loop1), and the tubulin-FtsZ signature G-box motif(SAG)GGTG(SAT)G (loop3). A simple working model envisages loop1 and loop3together at the nucleotide binding site, while loops 2 and 4 are at thesurface of the protein, in agreement with proteolytic and antigenicaccessibility results in tubulin. The model is compatible with studies oftubulin and FtsZ mutants. It is proposed that this region constitutes acommon structural and evolutionary nucleus of tubulins and FtsZ which isdifferent from typical GTPases.

• Nogales E, Wolf SG, Khan IA, Luduena RF, Downing KH
• Structure of tubulin at 6.5 A and location of the taxol-binding site.
• Nature. 1995; 375: 424-7
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• Tubulin, the major component of microtubules, is a heterodimer of twochains, alpha and beta, both of relative molecular mass 50,000 (Mr50K) andwith 40-50% identity. The isotypic variety and conformational flexibilityof tubulin have so far made it impossible to obtain crystals for X-raywork. Structural knowledge of tubulin has been limited to about 20 A fromX-ray diffraction of oriented microtubules, and from electron microscopyof microtubules and zinc-induced crystalline sheets in negative stain. Thesheets consist of protofilaments similar to those in microtubules butassociated in an antiparallel arrangement, and their two-dimensionalcharacter is ideal for high-resolution electron microscopy. Here wepresent a three-dimensional reconstruction of tubulin to 6.5 A resolution,obtained by electron crystallography of zinc-induced two-dimensionalcrystals of the protein. The alpha- and beta-subunits appear topologicallysimilar, in agreement with their sequence homology. Several features canbe defined in terms of secondary structure. An apparent alpha-helicalportion, adjacent to both interdimer and inter-protofilament contacts, istentatively attributed to a segment near the carboxy terminus of theprotein. We can assign the alpha- and beta-subunits on the basis ofprojection studies of the binding of taxol, which show one taxol site pertubulin heterodimer, in agreement with the known stoichiometry of taxol inmicrotubules. These studies indicate that taxol affects the interactionbetween protofilaments; to our knowledge, this is the first time that aligand-binding site has been visualized in the tubulin molecule.