Straight Protofilaments Research Articles

Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6–3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic. We also determined a crystal structure of microtubule-unbound CaKip3-ADP (2.0 Å) and analyzed the biochemical activity of CaKip3 and kinesin-1 mutants. These data reveal that the microtubule depolymerization activity of kinesin-8 originates from conformational changes of its motor core that are amplified by dynamic contacts between its extended loop-2 and tubulin. On curved microtubule ends, loop-1 inserts into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin and assist depolymerization. On straight tubulin protofilaments in the microtubule lattice, loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. We propose that these tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disassembling Microtubule Tips.

Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or RNA. Often the filamentous substrates serve merely as passive tracks for mechanoenzymes but microtubules and F-actin are themselves dynamic protein polymers, capable of generating movement and force independently of conventional motors. Microtubule-driven forces are particularly important during mitosis, when they align duplicated chromosomes at the metaphase plate and then pull them apart during anaphase. These vital movements depend on specialized protein assemblies called kinetochores that couple the chromosomes to the tips of dynamic microtubule filaments, thereby allowing filament shortening to produce pulling forces. Although great strides have been made toward understanding the structures and functions of many kinetochore subcomplexes, the biophysical basis for their coupling to microtubule tips remains unclear. During tip disassembly, strain energy is released when straight protofilaments in the microtubule lattice curl outward, creating a conformational wave that propagates down the microtubule. A popular viewpoint is that the protofilaments as they curl outward hook elements of the kinetochore and tug on them, transferring some of their curvature strain energy to the kinetochore. As a first step toward testing this idea, we recently developed a laser trap assay to directly measure the working strokes generated by curling protofilaments. Our "wave" assay is based on an earlier pioneering study, with improvements that allow measurement of curl-driven movements as functions of force and quantification of their conformational strain energy. In this chapter, we provide a detailed protocol for our assay and describe briefly our instrument setup and data analysis methods.

Nanoscopic Structural Fluctuations of Disassembling Microtubules Revealed by Label-Free Super-Resolution Microscopy.

Microtubules are cytoskeletal polymers of tubulin dimers assembled into protofilaments that constitute nanotubes undergoing periods of assembly and disassembly. Static electron micrographs suggest a structural transition of straight protofilaments into curved ones occurring at the tips of disassembling microtubules. However, these structural transitions have never been observed and the process of microtubule disassembly thus remains unclear. Here, label-free optical microscopy capable of selective imaging of the transient structural changes of protofilaments at the tip of a disassembling microtubule is introduced. Upon induced disassembly, the transition of ordered protofilaments into a disordered conformation is resolved at the tip of the microtubule. Imaging the unbinding of individual tubulin oligomers from the microtubule tip reveals transient pauses and relapses in the disassembly, concurrent with increased organization of protofilament segments at the microtubule tip. These findings show that microtubule disassembly is a discrete process and suggest a stochastic mechanism of switching from the disassembly to the assembly phase.

Mechanistic Insights into Plant Chiral Growth

The latent left–right asymmetry (chirality) of vascular plants is best witnessed as a helical elongation of cylindrical organs in climbing plants. Interestingly, helical handedness is usually fixed in given species, suggesting genetic control of chirality. Arabidopsis thaliana, a small mustard plant, normally does not twist but can be mutated to exhibit helical growth in elongating organs. Genetic, molecular and cell biological analyses of these twisting mutants are providing mechanistic insights into the left–right handedness as well as how potential organ skewing is suppressed in most plants. Growth direction of elongating plant cells is determined by alignment of cellulose microfibrils in cell walls, which is guided by cortical microtubules localized just beneath the plasma membrane. Mutations in tubulins and regulators of microtubule assembly or organization give rise to helical arrangements of cortical microtubule arrays in Arabidopsis cells and cause helical growth of fixed handedness in axial organs such as roots and stems. Whether tubulins are assembled into a microtubule composed of straight or tilted protofilaments might determine straight or twisting growth. Mechanistic understanding of helical plant growth will provide a paradigm for connecting protein filament structure to cellular organization.

Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli.

FtsZ, a tubulin-like GTPase, is essential for bacterial cell division. In the presence of GTP, FtsZ polymerizes into filamentous structures, which are key to generating force in cell division. However, the structural basis for the molecular mechanism underlying FtsZ function remains to be elucidated. In this study, crystal structures of the enzymatic domains of FtsZ from Klebsiella pneumoniae (KpFtsZ) and Escherichia coli (EcFtsZ) were determined at 1.75 and 2.50 Å resolution, respectively. Both FtsZs form straight protofilaments in the crystals, and the two structures adopted relaxed (R) conformations. The T3 loop, which isinvolved in GTP/GDP binding and FtsZ assembly/disassembly, adopted a unique open conformation in KpFtsZ, while the T3 loop of EcFtsZ was partially disordered. The crystal structure of EcFtsZ can explain the results from previous functional analyses using EcFtsZ mutants.

Assembly properties of the bacterial tubulin homolog FtsZ from the cyanobacterium Synechocystis sp. PCC 6803

The tubulin homolog FtsZ is the major cytoskeletal protein in the bacterial cell division machinery, conserved in almost all bacteria, archaea, and chloroplasts. Bacterial FtsZ assembles spontaneously into single protofilaments, sheets, and bundles in vitro, and it also accumulates at the site of division early during cell division, where it forms a dynamic protein complex, the contractile ring or Z-ring. The biochemical properties of FtsZ proteins from many bacteria have been studied, but comparable insights into FtsZs from cyanobacteria are limited. Here, using EM and light-scattering assays, we studied the biochemical and assembly properties of SyFtsZ, the FtsZ protein from the cyanobacterial strain Synechocystis sp. PCC 6803. SyFtsZ had a slow GTPase activity of ∼0.4 GTP/FtsZ molecule/min and assembled into thick, straight protofilament bundles and curved bundles, designated toroids. The assembly of SyFtsZ in the presence of GTP occurred in two stages. The first stage consisted of the assembly of single-stranded straight protofilaments and opened circles; in the second stage, the protofilaments associated into straight protofilament bundles and toroids. In addition to these assemblies, we also observed highly curved oligomers and minirings after GTP hydrolysis or in the presence of excess GDP. The three types of protofilaments of SyFtsZ observed here provide support for the hypothesis that a constriction force due to curved protofilaments bends the membrane. In summary, our findings indicate that, unlike other bacterial FtsZ, SyFtsZ assembles into thick protofilament bundles. This bundling is similar to that of chloroplast FtsZ, consistent with its origin in cyanobacteria.

Mechanical properties of tubulin intra- and inter-dimer interfaces and their implications for microtubule dynamic instability.

Thirteen tubulin protofilaments, made of αβ-tubulin heterodimers, interact laterally to produce cytoskeletal microtubules. Microtubules exhibit the striking property of dynamic instability, manifested in their intermittent growth and shrinkage at both ends. This behavior is key to many cellular processes, such as cell division, migration, maintenance of cell shape, etc. Although assembly and disassembly of microtubules is known to be linked to hydrolysis of a guanosine triphosphate molecule in the pocket of β-tubulin, detailed mechanistic understanding of corresponding conformational changes is still lacking. Here we take advantage of the recent generation of in-microtubule structures of tubulin to examine the properties of protofilaments, which serve as important microtubule assembly and disassembly intermediates. We find that initially straight tubulin protofilaments, relax to similar non-radially curved and slightly twisted conformations. Our analysis further suggests that guanosine triphosphate hydrolysis primarily affects the flexibility and conformation of the inter-dimer interface, without a strong impact on the shape or flexibility of αβ-heterodimer. Inter-dimer interfaces are significantly more flexible compared to intra-dimer interfaces. We argue that such a difference in flexibility could be key for distinct stability of the plus and minus microtubule ends. The higher flexibility of the inter-dimer interface may have implications for development of pulling force by curving tubulin protofilaments during microtubule disassembly, a process of major importance for chromosome motions in mitosis.

Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule.

Microtubule (MT) is made of αβ-tubulin heterodimers that dynamically assemble into a hollow nanotube composed of straight protofilaments. MT dynamics is facilitated by hydrolysis of guanosine-5'-triphosphate (GTP) and can be inhibited by either anticancer agents like taxol or the nonhydrolyzable GTP analogues like GMPPCP. Using high-resolution synchrotron X-ray scattering, we have measured and analyzed the scattering curves from solutions of dynamic MT (in other words, in the presence of excess GTP and free of dynamic-inhibiting agents) and examined the effect of two MT stabilizers: taxol and GMPPCP. Previously, we have analyzed the structure of dynamic MT by docking the atomic model of tubulin dimer onto a 3-start left handed helical lattice, derived from the PDB ID 3J6F . 3J6F corresponds to a MT with 14 protofilaments. In this paper, we took into account the possibility of having MT structures containing between 12 and 15 protofilaments. MTs with 12 protofilaments were never observed. We determined the radii, the pitch, and the distribution of protofilament number that best fit the scattering data from dynamic MT or stabilized MT by taxol or GMPPCP. We found that the protofilament number distribution shifted when the MT was stabilized. Taxol increased the mass fraction of MT with 13 protofilaments and decreased the mass fraction of MT with 14 protofilaments. GMPPCP reduced the mass fraction of MT with 15 protofilaments and increased the mass fraction of MT with 14 protofilaments. The pitch, however, remained unchanged regardless of whether the MT was dynamic or stabilized. Higher tubulin concentrations increased the fraction of dynamic MT with 14 protofilaments.

Paclitaxel suppresses Tau-mediated microtubule bundling in a concentration-dependent manner

BackgroundMicrotubules (MTs) are protein nanotubes comprised of straight protofilaments (PFs), head to tail assemblies of αβ-tubulin heterodimers. Previously, it was shown that Tau, a microtubule-associated protein (MAP) localized to neuronal axons, regulates the average number of PFs in microtubules with increasing inner radius <RinMT> observed for increasing Tau/tubulin-dimer molar ratio ΦTau at paclitaxel/tubulin-dimer molar ratio ΛPtxl=1/1. MethodsWe report a synchrotron SAXS and TEM study of the phase behavior of microtubules as a function of varying concentrations of paclitaxel (1/32≤ΛPtxl≤1/4) and Tau (human isoform 3RS, 0≤Φ3RS≤1/2) at room temperature. ResultsTau and paclitaxel have opposing regulatory effects on microtubule bundling architectures and microtubule diameter. Surprisingly and in contrast to previous results at ΛPtxl=1/1 where microtubule bundles are absent, in the lower paclitaxel concentration regime (ΛPtxl≤1/4), we observe both microtubule doublets and triplets with increasing Tau. Furthermore, increasing paclitaxel concentration (up to ΛPtxl=1/1) slightly decreased the average microtubule diameter (by ~1 PF) while increasing Tau concentration (up to Φ3RS=1/2) significantly increased the diameter (by ~2–3 PFs). ConclusionsThe suppression of Tau-mediated microtubule bundling with increasing paclitaxel is consistent with paclitaxel seeding more, but shorter, microtubules by rapidly exhausting tubulin available for polymerization. Microtubule bundles require the aggregate Tau-Tau attractions along the microtubule length to overcome individual microtubule thermal energies disrupting bundles. General significanceInvestigating MAP-mediated interactions between microtubules (as it relates to in vivo behavior) requires the elimination or minimization of paclitaxel.

Super-Resolution Imaging of Slowly Depolymerizing Microtubules Reveals No Curved Protofilaments with a Lifetime Longer than Seconds

Microtubules are 25 nm diameter, micrometer long, cylindrical protein polymers involved in cell division, cell organization, and intracellular transport among other roles. Microtubule dynamics, that is, how microtubules grow and shrink (polymerization, catastrophe, depolymerization, and rescue) plays a role in each of these functions. Microtubule depolymerization (shrinkage), in particular, is proposed to involve a structural transition from straight protofilaments during microtubule polymerization to curled protofilaments at microtubule plus-ends during shrinkage. Direct evidence of this curved structure (sometimes called ‘ram's horns’) comes from static electron microscopy images and from unresolved intensity increases in light microscopy. As of now, however, no dynamic images of depolymerizing microtubules have directly resolved this ram's horns structure.We use STORM super-resolution microscopy on a custom-built microscope (the Warwick Open Source Microscope, WOSM) to image microtubules, in vitro, with a spatial resolution better than 20 nm and a temporal resolution of 10 seconds. We slow microtubule depolymerization to a similar timescale using sub-stoichiometric concentrations of paclitaxel or kinesin binding in rigor. Movies of these slowly depolymerizing microtubules show no curved protofilaments at microtubule plus ends. Instead, depolymerizing microtubules remain straight objects with a diameter of 25 nm. Our interpretation of these negative results are that the ram's horns structures have a lifetime shorter than seconds. An alternative hypothesis is that partial stabilization of microtubules is concomitant with a straightening of protofilaments at the shrinking end. This alternative suggests that proposed coupling mechanisms for force generation by depolymerizing microtubules must accommodate straight, rather than curved, protofilaments.

Tau-ism: The Yin and Yang of Microtubule Sliding, Detachment, and Rupture

Traumatic brain injury is a nondege-nerative, noncongenital insult to thebrain caused by external mechanicalforces. Recent studies suggest thateven mild concussions—if repeti-tive—can trigger progressive neuro-logical degeneration, a condition thatis now widely known as ‘‘chronic trau-matic encephalopathy’’ (1). Notably,chronic traumatic encephalopathycon-tinues to progress even decades afterthe initial insult; the more severe theoriginal injury and the longer thesurvival, the greater the severity ofneurodegeneration. Progressive axonaldamage and structural degradationare classic hallmarks of chronic trau-matic encephalopathy. Strikingly,these symptoms appear to be sharedby a number of other neurodegenera-tive diseases including Alzheimer’sdisease and Parkinsonism. However,the molecular mechanisms of axonalfailure remain poorly understood.One of the most common patholog-ical features of traumatic brain injuryis diffuse axonal injury (2). To date,the only method to reliably diagnosediffuse axonal injury is post mortemhistopathology, where it manifests it-self through extensive damage inwhite-matter tissue. More than half acentury ago, experiments with gelatinmolds have established that patholog-ical white-matter damage is a directconsequence of elevated mechanicalstrains and strain rates (3): At lowstrain rates, axons that make upwhite-matter tissue are highly com-pliant and ductile; they can easilydeform and revert to their initialconformation. At high strain rates,axons stiffen and become brittle; theyare vulnerable to mechanical failure(Fig. 1). Axonal failure manifests itselfin two modes, primary axotomy,the immediate, complete mechanicalrupture, and secondary axotomy, theprogressive degradation and gradualfailure (1). Although secondary axot-omy is by far the more common failuremode, the precise sequence of eventsby which the axonal cytoskeleton de-grades is unknown.The axonal cytoskeleton is made upof microtubules, neurofilaments, andmicrofilaments. Neuronal microtu-bules are structurally similar to micro-tubules in all other cells of our body:Composed of heterodimers of a- andb-tubulin that form 13 laterally joinedprotofilaments, microtubules are hol-low tubes with a diameter of 24 nm.With a high resistance to bending anda stiffness of 2.0 GPa, microtubulesareundoubtedlythestrongestcytoskel-etal filaments in eukaryotic cells. Assuch, they play a unique role in a num-ber of cellular processes, maintainingstructural stability and providinghighways for intracellular transport.While the microtubule ultrastructureissimilarinall eukaryoticcells,micro-tubule organization in neurons differssignificantly from nonneuronal cells(4): Axons can extend up to a meterin length and their microtubules neverrun continuously from the cell bodyto the distal end. Instead, they formbundles of microtubule segments with10–20 microtubules in any given crosssection. Neuronal microtubules arenucleated at the centrosome withinthe cell body, then rapidly released,and delivered into the axon via molec-ular motors.Duringtransport,microtu-bules shorten to provide subunits forthe elongation of other microtubules.This explains why the microtubulelength can vary significantly along theaxon, with up to 100 mm and longertoward the cell body and 2 mm andshorter toward the distal end (3). Indi-vidual microtubules are stabilized andcross-linked to form the axonal cyto-skeleton via microtubule-associatedproteins.The most prominent microtubule-associated protein, tau, was discoveredmore than four decades ago (5). Adecade later, tau took center stage asthe major component of neurofibrillarytangles, which are now widely knownas the primary markers of Alzheimer’sdisease (6). Tau is primarily a neuronalprotein.Intheadulthumanbrain,alter-native mRNA splicing generates sixmajor isoforms of tau varying from352 to 441 amino acids in length. Allisoforms share three common do-mains: a microtubule-binding domaincomposed of repeats of an evolution-arily conserved tubulin binding motif,a positively charged proline-rich re-gion, and a negatively charged N-ter-minus (7). The six isoforms differ bythe number of microtubule-binding re-peats, either three or four, and by thepresence or absence of one or twoN-terminal inserts. Tau is nativelydisordered. By binding to microtu-bules, tau becomes more organizedand contributes directly or indirectlyto key structural and regulatorycellular function. Within individualmicrotubules, tau modulates microtu-bule polymerization, controls microtu-bule structure, and regulates axonaltransport; the tubulin-binding repeatsof tau bind to hydrophobic pocketsbetween the a- and b-tubulin hetero-dimers of a microtubule to stabilizeits straight protofilament conforma-tion (8). Within the axonal cytoskel-eton, tau promotes the assembly ofindividual microtubules into well-organized, evenly spaced bundles;two tau proteins of neighboring micro-tubules form an electrostatic zipper

The subtle allostery of microtubule dynamics.

New high-resolution structures of microtubules reveal that GTP and taxol each stimulate microtubule assembly by inducing straight protofilaments and promoting extension of the interdimer spacing. However, these effects take place through different mechanisms: GTP directly extends loops around the active site, whereas taxol works like a remote lever.

Microtubules in Bacteria: Ancient Tubulins Build a Five-Protofilament Homolog of the Eukaryotic Cytoskeleton

Author SummaryBacteria are generally distinguished from the cells of fungi, plants, and animals (eukaryotes) not only by their much smaller size but also by the absence of certain subcellular structures such as nuclei, internal organelles, and microtubules. Using state-of-the-art microscopy, we demonstrate here that microtubules do exist in some bacteria. These bacterial microtubules are built from proteins that are closely related to the microtubule proteins in eukaryotes. Bacterial microtubules are smaller in diameter than their counterparts in eukaryotic cells but have the same basic architecture. We propose that bacterial microtubules represent primordial structures that preceded eukaryotic microtubules evolutionarily. Because bacterial microtubules can be produced and handled in the lab more easily than their eukaryotic counterparts, they may become useful tools for microtubule research and anti-cancer drug screening.

Acting Like Actin

Biochemistry In a dividing cell, DNA must be replicated and accurately partitioned between the two daughter cells. In eukaryotes, microtubules, which are linked to the centromeric DNA by kinetochores, separate the replicated chromosomes with the help of motor proteins. Prokaryotic plasmid-partitioning systems are similar in that they involve an adaptor protein that links DNA to a filament-forming protein; however, no motor proteins are involved, and nucleotidehydrolysis dependent filament dynamics are responsible for movement. To gain insight into the segregation of the Bacillus thuringiensis pBToxis plasmid, Aylett et al. determined the structure of two protofilaments of the tubulin/FtsZ-like protein, TubZ, which mediates plasmid segregation. A combination of crystallography and electron microscopy revealed that instead of forming tubulin-like straight protofilaments that organize into microtubules, TubZ forms twisted double filaments that can bundle, reminiscent of structures formed by ParM, an actin-like prokaryotic partitioning filament. That two disparate proteins have evolved to perform a similar function using similar superstructures suggests strong evolutionary constraints on partitioning systems. Proc. Natl. Acad. Sci. U.S.A. 107 , 19766 (2010).

Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional β-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from β-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

Structural changes occur in the alphabeta-tubulin heterodimer during the microtubule assembly/disassembly cycle. Their most prominent feature is a transition from a straight, microtubular structure to a curved structure. There is a broad range of small molecule compounds that disturbs the microtubule cycle, a class of which targets the colchicine-binding site and prevents microtubule assembly. This class includes compounds with very different chemical structures, and it is presently unknown whether they prevent tubulin polymerization by the same mechanism. To address this issue, we have determined the structures of tubulin complexed with a set of such ligands and show that they interfere with several of the movements of tubulin subunits structural elements upon its transition from curved to straight. We also determined the structure of tubulin unliganded at the colchicine site; this reveals that a beta-tubulin loop (termed T7) flips into this site. As with colchicine site ligands, this prevents a helix which is at the interface with alpha-tubulin from stacking onto a beta-tubulin beta sheet as in straight protofilaments. Whereas in the presence of these ligands the interference with microtubule assembly gets frozen, by flipping in and out the beta-subunit T7 loop participates in a reversible way in the resistance to straightening that opposes microtubule assembly. Our results suggest that it thereby contributes to microtubule dynamic instability.

Bacterial Division: Another Way to Box in the Ring

Proper placement of the cell division site in some rod-shaped bacteria requires two different negative regulatory systems, nucleoid occlusion and the Min proteins. Caulobacter crescentus lacks these systems, but recent work has uncovered a novel regulator that achieves the same goals.

Nucleotide-dependent bending flexibility of tubulin regulates microtubule assembly

The atomic structure of tubulin in a polymerized, straight protofilament is clearly distinct from that in a curved conformation bound to a cellular depolymerizer. The nucleotide contents are identical, and in both cases the conformation of the GTP-containing, intra-dimer interface is indistinguishable from the GDP-containing, inter-dimer contact. Here we present two structures corresponding to the start and end points in the microtubule polymerization and hydrolysis cycles that illustrate the consequences of nucleotide state on longitudinal and lateral assembly. In the absence of depolymerizers, GDP-bound tubulin shows distinctive intra-dimer and inter-dimer interactions and thus distinguishes the GTP and GDP interfaces. A cold-stable tubulin polymer with the non-hydrolysable GTP analogue GMPCPP, containing semi-conserved lateral interactions, supports a model in which the straightening of longitudinal interfaces happens sequentially, starting with a conformational change after GTP binding that straightens the dimer enough for the formation of lateral contacts into a non-tubular intermediate. Closure into a microtubule does not require GTP hydrolysis.

Mutants of FtsZ Targeting the Protofilament Interface: Effects on Cell Division and GTPase Activity

The bacterial cell division protein FtsZ assembles into straight protofilaments, one subunit thick, in which subunits appear to be connected by identical bonds or interfaces. These bonds involve the top surface of one subunit making extensive contact with the bottom surface of the subunit above it. We have investigated this interface by site-directed mutagenesis. We found nine bottom and eight top mutants that were unable to function for cell division. We had expected that some of the mutants might poison cell division substoichiometrically, but this was not found for any mutant. Eight of the bottom mutants exhibited dominant negative effects (reduced colony size) and four completely blocked colony formation, but this required expression of the mutant protein at four to five times the wild-type FtsZ level. Remarkably, the top mutants were even weaker, most showing no effect at the highest expression level. This suggests a directional assembly or treadmilling, where subunit addition is primarily to the bottom end of the protofilament. Selected pairs of top and bottom mutants showed no GTPase activity up to 10 to 20 microM, in contrast to the high GTPase activity of wild-type FtsZ above 1 muM. Overall, these results suggest that in order for a subunit to bind a protofilament at the 1 microM K(d) for elongation, it must have functional interfaces at both the top and bottom. This is inconsistent with the present model of the protofilament, as a simple stack of subunits one on top of the other, and may require a new structural model.

Structural rearrangements in tubulin following microtubule formation.

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.