Tubulin Tail Research Articles

Structural and Molecular Basis for Katanin-Mediated Severing of Glutamylated Microtubules

Katanin was the first microtubule (MT)-severing enzyme discovered, but how katanin executes MTsevering remains poorly understood. Here, we report X-ray crystal structures of the apo and ATPγS-bound states of the catalytic AAA domain of human katanin p60 at 3.0 and 2.9Å resolution, respectively. Comparison of the two structures reveals conformational changes induced by ATP binding and how such changes ensure hexamer stability. Moreover, we uncover structural details of pore loops (PLs) and show that Arg283, a residue unique to katanin among MT-severing enzymes, protrudes from PL1 and lines the entry of the catalytic pore. Functional studies suggest that PL1 and Arg283 play essential roles in the recognition and remodeling of the glutamylated, C-terminal tubulin tail and regulation of axon growth. In addition, domain-swapping experiments in katanin and spastin suggest that the non-homologous N-terminal region, which contains the MT-interacting and trafficking domain and a linker, confers specificity to the severing process.

Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia

Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a "tubulin code" that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C.elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliaryspecialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C.elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported byciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release inaliving animal. We suggest that cell-specific controlof MT glutamylation may be a conserved mechanism to specialize the form and function ofcilia.

Crystal structure of tubulin tyrosine ligase-like 3 reveals essential architectural elements unique to tubulin monoglycylases

Glycylation and glutamylation, the posttranslational addition of glycines and glutamates to genetically encoded glutamates in the intrinsically disordered tubulin C-terminal tails, are crucial for the biogenesis and stability of cilia and flagella and play important roles in metazoan development. Members of the diverse family of tubulin tyrosine ligase-like (TTLL) enzymes catalyze these modifications, which are part of an evolutionarily conserved and complex tubulin code that regulates microtubule interactions with cellular effectors. The site specificity of TTLL enzymes and their biochemical interplay remain largely unknown. Here, we report an in vitro characterization of a tubulin glycylase. We show that TTLL3 glycylates the β-tubulin tail at four sites in a hierarchical order and that TTLL3 and the glutamylase TTLL7 compete for overlapping sites on the tubulin tail, providing a molecular basis for the anticorrelation between glutamylation and glycylation observed in axonemes. This anticorrelation demonstrates how a combinatorial tubulin code written in two different posttranslational modifications can arise through the activities of related but distinct TTLL enzymes. To elucidate what structural elements differentiate TTLL glycylases from glutamylases, with which they share the common TTL scaffold, we determined the TTLL3 X-ray structure at 2.3-Å resolution. This structure reveals two architectural elements unique to glycyl initiases and critical for their activity. Thus, our work sheds light on the structural and functional diversification of TTLL enzymes, and constitutes an initial important step toward understanding how the tubulin code is written through the intersection of activities of multiple TTLL enzymes.

Molecular interactions between tubulin tails and glutamylases reveal determinants of glutamylation patterns.

Posttranslational modifications of tubulin currently emerge as key regulators of microtubule functions. Polyglutamylation generates a variety of modification patterns that are essential for controlling microtubule functions in different cell types and organelles, and deregulation of these patterns has been linked to ciliopathies, cancer and neurodegeneration. How the different glutamylating enzymes determine precise modification patterns has so far remained elusive. Using computational modelling, molecular dynamics simulations and mutational analyses we now show how the carboxy-terminal tails of tubulin bind into the active sites of glutamylases. Our models suggest that the glutamylation sites on α- and β-tubulins are determined by the positioning of the tails within the catalytic pocket. Moreover, we found that the binding modes of α- and β-tubulin tails are highly similar, implying that most enzymes could potentially modify both, α- and β-tubulin. This supports a model in which the binding of the enzymes to the entire microtubule lattice, but not the specificity of the C-terminal tubulin tails to their active sites, determines the catalytic specificities of glutamylases.

Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes.

Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.

The Role of the C-Terminal Tails of Tubulin in Microtubule Dynamics

The Role of the C-Terminal Tails of Tubulin in Microtubule Dynamics

Molecular Determinants of Tubulin's C-Terminal Tail Conformational Ensemble.

Tubulin is important for a wide variety of cellular processes including cell division, ciliogenesis, and intracellular trafficking. To perform these diverse functions, tubulin is regulated by post-translational modifications (PTM), primarily at the C-terminal tails of both the α- and β-tubulin heterodimer subunits. The tubulin C-terminal tails are disordered segments that are predicted to extend from the ordered tubulin body and may regulate both intrinsic properties of microtubules and the binding of microtubule associated proteins (MAP). It is not understood how either interactions with the ordered tubulin body or PTM affect tubulin's C-terminal tails. To probe these questions, we developed a method to isotopically label tubulin for C-terminal tail structural studies by NMR. The conformational changes of the tubulin tails as a result of both proximity to the ordered tubulin body and modification by mono- and polyglycine PTM were determined. The C-terminal tails of the tubulin dimer are fully disordered and, in contrast with prior simulation predictions, exhibit a propensity for β-sheet conformations. The C-terminal tails display significant chemical shift differences as compared to isolated peptides of the same sequence, indicating that the tubulin C-terminal tails interact with the ordered tubulin body. Although mono- and polyglycylation affect the chemical shift of adjacent residues, the conformation of the C-terminal tail appears insensitive to the length of polyglycine chains. Our studies provide important insights into how the essential disordered domains of tubulin function.

Tubulin Tails are Intrinsically Disordered Polyanions that Regulate Binding to other Proteins by Sequence as Well as Charge

Microtubules (MT) are built by polymerization of the alpha/beta tubulin heterodimer. The two tubulins are very similar to each other and are composed of a globular body containing about 95% of the protein mass and an unstructured C-terminal tail (CTT) that contains about 40% of the protein's net negative charge. The CTT is the locus of most sequence differences between tubulin isotypes and also the target of most posttranslational modifications (PTMs). The tails extend from the tubulin body and from the outside surface of MT, and are often the first part of tubulin / MT that other proteins encounter. The high charge density on the tails can promote or oppose interaction interactions with other proteins. Surprisingly, perhaps, interactions with some proteins requires the negative charges, but are still highly modulated by sequence differences between tails from different isotypes. PTMs can add an additional, significant modulation of binding that can be determinative. Specific examples of each of these will be presented and discussed in the context of: Intrinsically Disordered Tails are Not Just Charged Strings.

Chromokinesins NOD and KID use Alternative Nucleotide Mechanisms and One-Dimensional Diffusion to Target Microtubule Plus Ends

Chromokinesins have been found to associate with chromosomes during cell division and play a variety of functions including chromosome positioning, chromosome condensation, spindle bipolarity, and cytokinesis. These motors are thought to generate the elusive polar ejection force that drives chromatin away from the spindle poles. Kinesin-10's are chromokinesins that include human KID (also known as KIF22) and Drosophila melanogaster NOD, which share an N-terminal kinesin-like catalytic domain, a central stalk domain, and a C-terminal DNA-binding domain. Due to their common in vivo physiologies, we propose that kinesin-10's work together in teams of motors to translocate/target to spindle microtubule plus-ends. Using steady-state kinetics, we have characterized the motor domain constructs of NOD and KID by measuring the basal, unpolymerized tubulin-stimulated, and MT-stimulated steady-state ATPase and GTPase kinetics under both low (∼21 mM) and physiologically relevant (∼111 mM) ionic strength conditions. These results suggest both purine nucleotides may be utilized by kinesin-10's in vivo and set the foundation for comparing the ATPase and GTPase mechanisms of these two unconventional kinesins. Using GFP-tagged kinesin-10's and total internal reflection fluorescence (TIRF) microscopy, we observed the microtubule-bound NOD either remaining stationary or engaging in a one-dimensional random walk across the microtubule lattice. These two modes of microtubule binding are interchangeable and movement along the microtubule is rapid (similar to kinesin-13 and other microtubule end-binding proteins) and without directionality preference. Under these conditions, we observe significantly higher incidence of microtubule end-binding events, which is increased after removal of the negatively-charge C-terminal tails of tubulin using subtilisin. Our continued studies reveal mechanistic and functional details of kinesin-10 force generation and therefore provide new insights into the molecular motion that drives the polar ejection force during cell division.

Studying the Effects of Modified Tubulin C-Terminal Tails on Katanin Severing Activity

Microtubule severing enzymes are AAA+ enzymes that sever microtubule filaments in order to regulate the organization of the cytoskeleton of cells. We have recently shown that katanin, the first identified sever enzyme, is inhibited by free tubulin dimers. Further, we showed that inhibition is specific for the carboxy-terminal tail and is isotype and post-translationally specific. Further, the inhibition of katanin is more potent than has been observed for spastin, a second severing enzyme, and has different reactions to different isotypes and post-translational modifications. This implies that katanin is regulated through different paths than spastin. We are currently investigation the inhibitory effects of numerous carboxy-terminal tail types. We perform in vitro reconstitution assays using wholly purified proteins and polypeptide carboxy-terminal tails of tubulin. This work provides a view into how katanin recognition of the CTT of tubulin regulates microtubule severing. A further understanding of this interaction is needed in order to understand how microtubule regulation is performed at the molecular level.

Tubulin Tail Sequences and Post-translational Modifications Regulate Closure of Mitochondrial Voltage-dependent Anion Channel (VDAC)

It was previously shown that tubulin dimer interaction with the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) blocks traffic through the channel and reduces oxidative metabolism and that this requires the unstructured anionic C-terminal tail peptides found on both α- and β-tubulin subunits. It was unclear whether the α- and β-tubulin tails contribute equally to VDAC blockade and what effects might be due to sequence variations in these tail peptides or to tubulin post-translational modifications, which mostly occur on the tails. The nature of the contribution of the tubulin body beyond acting as an anchor for the tails had not been clarified either. Here we present peptide-protein chimeras to address these questions. These constructs allow us to easily combine a tail peptide with different proteins or combine different tail peptides with a particular protein. The results show that a single tail grafted to an inert protein is sufficient to produce channel closure similar to that observed with tubulin. We show that the β-tail is more than an order of magnitude more potent than the α-tail and that the lower α-tail activity is largely due to the presence of a terminal tyrosine. Detyrosination activates the α-tail, and activation is reversed by the removal of the glutamic acid penultimate to the tyrosine. Nitration of tyrosine reverses the tyrosine inhibition of binding and even induces prolonged VDAC closures. Our results demonstrate that small changes in sequence or post-translational modification of the unstructured tails of tubulin result in substantial changes in VDAC closure.

Microtubule C-Terminal Tails Can Change Characteristics of Motor Force Production.

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties--if at all--is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin-1, and also a mitotic kinesin likely optimized for group function, kinesin-5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin-1 functioned similarly on the two sets of MTs--in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin-1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin-5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for kinesin-5 on the cancer-cell MTs, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar.

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

The microtubule-associated protein, TPX2, regulates the activity of the mitotic kinesin, Eg5, but the mechanism of regulation is not established. Using total internal reflection fluorescence microscopy, we observed that Eg5, in extracts of mammalian cells expressing Eg5-EGFP, moved processively toward the microtubule plus-end at an average velocity of 14 nm/s. TPX2 bound to microtubules with an apparent dissociation constant of ∼ 200 nm, and microtubule binding was not dependent on the C-terminal tails of tubulin. Using single molecule assays, we found that full-length TPX2 dramatically reduced Eg5 velocity, whereas truncated TPX2, which lacks the domain that is required for the interaction with Eg5, was a less effective inhibitor at the same concentration. To determine the region(s) of Eg5 that is required for interaction with TPX2, we performed microtubule gliding assays. Dimeric, but not monomeric, Eg5 was differentially inhibited by full-length and truncated TPX2, demonstrating that dimerization or residues in the neck region are important for the interaction of TPX2 with Eg5. These results show that both microtubule binding and interaction with Eg5 contribute to motor inhibition by TPX2 and demonstrate the utility of mammalian cell extracts for biophysical assays.

Stability Analysis of Jacobi Elliptic Solutions of Microtubule

A nonlinear dynamic model which elucidate the mechanism of the internal motion occuring during the assembly of microtubules (MTs) is considered. The nonlinear space-time dynamics, defined in terms of celebrated ‘solitonic’ equations, brings indispensable tools for understanding, prediction and control of complex behaviors in both physical and life sciences. MTs is assumed to be a single longitudinal degree of freedom per tubulin dimer, and the motion equation is reduced to the discrete nonlinear Schr ¨ odinger equation. Exact solutions are investigated using the Jacobian elliptic fonctions approach. It is shown that such a nonlinear model can lead to the existence of kink solitons, breather as well as Jacobi solutions which can propagate along the microtubule outer surface, and the tubulin tail soliton collisions could serve as elementary computational gates that control cytoskeletal processes. We examine the stability of these solutions and found that the kink solution is stable with respect to small-amplitude fluctuations.

The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids.

The posttranslational modification of carboxy-terminal tails of tubulin plays an important role in the regulation of the microtubule cytoskeleton. Enzymes responsible for deglutamylating tubulin have been discovered within a novel family of mammalian cytosolic carboxypeptidases. The discovery of these enzymes also revealed the existence of a range of other substrates that are enzymatically deglutamylated. Only four of six mammalian cytosolic carboxypeptidases had been enzymatically characterized. Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency. Deaspartylation is a novel posttranslational modification that could, in conjunction with deglutamylation, broaden the range of potential substrates that undergo carboxy-terminal processing. In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues. The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

Tubulin tyrosine ligase - structural and functional studies

Microtubules are polymers essential for cell morphogenesis, cell division and intracellular transport. This polymer's basic building block is the α/β tubulin heterodimer, which associates head-to-tail and laterally to form the microtubule. Tubulin is subject to diverse, abundant and evolutionarily conserved post-translational modifications that mark subpopulations of microtubules. The highest density and variety of post-translational modifications are found in neurons or cilia. Not surprisingly, tubulin modification enzymes have been linked to human diseases including cancers and neurodegenerative disorders. We will present our recent work using a combination of X-ray crystallography, small angle X-ray scattering and functional assays to investigate the mechanism of tubulin tyrosine ligase (TTL). TTL catalyzes the ATP-dependent post-translational addition of a tyrosine to the C-terminal end of detyrosinated α-tubulin. The detyrosination/tyrosination cycle regulates recruitment of motors and proteins that track with the growing end of the microtubule. TTL function is essential for neuronal development and reduction in TTL levels is strongly associated with aggressive tumors resistant to chemotherapy. Our first X-ray crystal structure of TTL, defines the structural fold of the TTL-like family of tubulin-modifying enzymes. We show that TTL recognizes tubulin via a dual strategy: it engages the tubulin tail through low-affinity, high-specificity interactions through a conserved positively charged surface, and co-opts what is otherwise a homo-oligomerization interface in structurally related enzymes to form a tight hetero-oligomeric complex with tubulin. TTL forms an elongated complex with the tubulin dimer and prevents incorporation of the dimer into microtubules by capping the tubulin polymerization interface. Interestingly, TTL and stathmin, a ubiquitously expressed tubulin sequestering protein, compete for tubulin binding in vitro and stathmin inhibits tubulin tyrosination. These results suggest that TTL and stathmin have either a partially overlapping footprint on the tubulin dimer or that stathmin induces a tubulin conformation incompatible with stable TTL binding.

C-terminal region of MAP7 domain containing protein 3 (MAP7D3) promotes microtubule polymerization by binding at the C-terminal tail of tubulin.

MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0±0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics.

Microtubules Accelerate the Kinase Activity of Aurora-B by a Reduction in Dimensionality

Aurora-B is the kinase subunit of the Chromosome Passenger Complex (CPC), a key regulator of mitotic progression that corrects improper kinetochore attachments and establishes the spindle midzone. Recent work has demonstrated that the CPC is a microtubule-associated protein complex and that microtubules are able to activate the CPC by contributing to Aurora-B auto-phosphorylation in trans. Aurora-B activation is thought to occur when the local concentration of Aurora-B is high, as occurs when Aurora-B is enriched at centromeres. It is not clear, however, whether distributed binding to large structures such as microtubules would increase the local concentration of Aurora-B. Here we show that microtubules accelerate the kinase activity of Aurora-B by a “reduction in dimensionality.” We find that microtubules increase the kinase activity of Aurora-B toward microtubule-associated substrates while reducing the phosphorylation levels of substrates not associated to microtubules. Using the single molecule assay for microtubule-associated proteins, we show that a minimal CPC construct binds to microtubules and diffuses in a one-dimensional (1D) random walk. The binding of Aurora-B to microtubules is salt-dependent and requires the C-terminal tails of tubulin, indicating that the interaction is electrostatic. We show that the rate of Aurora-B auto-activation is faster with increasing concentrations of microtubules. Finally, we demonstrate that microtubules lose their ability to stimulate Aurora-B when their C-terminal tails are removed by proteolysis. We propose a model in which microtubules act as scaffolds for the enzymatic activity of Aurora-B. The scaffolding activity of microtubules enables rapid Aurora-B activation and efficient phosphorylation of microtubule-associated substrates.

ATP transport through VDAC and the VDAC-tubulin complex probed by equilibrium and nonequilibrium MD simulations.

Voltage-dependent anion channel (VDAC), the major channel of the mitochondrial outer membrane, serves as a principal pathway for ATP, ADP, and other respiratory substrates across this membrane. Using umbrella-sampling simulations, we established the thermodynamic and kinetic components governing ATP transport across the VDAC1 channel. We found that there are several low-affinity binding sites for ATP along the translocation pathway and that the main barrier for ATP transport is located around the center of the channel and is formed predominantly by residues in the N-terminus. The binding affinity of ATP to an open channel was found to be in the millimolar to micromolar range. However, we show that this weak binding increases the ATP translocation probability by about 10-fold compared with the VDAC pore in which attractive interactions were artificially removed. Recently, it was found that free dimeric tubulin induces a highly efficient, reversible blockage of VDAC reconstituted into planar lipid membranes. It was proposed that by blocking VDAC permeability for ATP/ADP and other mitochondrial respiratory substrates tubulin controls mitochondrial respiration. Using the Rosetta protein-protein docking algorithm, we established a tentative structure of the VDAC-tubulin complex. An extensive set of equilibrium and nonequilibrium (under applied electric field) molecular dynamics (MD) simulations was used to establish the conductance of the open and blocked channel. It was found that the presence of the unstructured C-terminal tail of tubulin in the VDAC pore decreases its conductance by more than 40% and switches its selectivity from anionic to cationic. The subsequent 1D potential of mean force (PMF) computations for the VDAC-tubulin complex show that the state renders ATP transport virtually impossible. A number of residues pivotal for tubulin binding to the channel were identified that help to clarify the molecular details of VDAC-tubulin interaction and to provide new insight into the mechanism of the control of mitochondria respiration by VDAC.