The solution structure of the N-terminal domain of human tubulin binding cofactor C reveals a platform for tubulin interaction

Human Tubulin Binding Cofactor C (TBCC) is a post-chaperonin involved in the folding and assembly of α- and β-tubulin monomers leading to the release of productive tubulin heterodimers ready to polymerize into microtubules. In this process it collaborates with other cofactors (TBC's A, B, D, and E) and forms a supercomplex with TBCD, β-tubulin, TBCE and α-tubulin. Here, we demonstrate that TBCC depletion results in multipolar spindles and mitotic failure. Accordingly, TBCC is found at the centrosome and is implicated in bipolar spindle formation. We also determine by NMR the structure of the N-terminal domain of TBCC. The TBCC N-terminal domain adopts a spectrin-like fold topology composed of a left-handed 3-stranded α-helix bundle. Remarkably, the 30-residue N-terminal segment of the TBCC N-terminal domain is flexible and disordered in solution. This unstructured region is involved in the interaction with tubulin. Our data lead us to propose a testable model for TBCC N-terminal domain/tubulin recognition in which the highly charged N-terminus as well as residues from the three helices and the loops interact with the acidic hypervariable regions of tubulin monomers.     

Tubulin Subunits Exist in an Activated Conformational State Generated and Maintained by Protein Cofactors

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.     

Possible regulation of microtubules through destabilization of tubulin

Chaperones and folding cofactors are known to mediate posttranslational folding of nascent tubulin, thus forming functional dimers. Based on sequence likeness, a novel protein similar to cofactor E, E-like protein (El), was identified. In overexpression experiments El, similar to a subset of folding factors (i.e. cofactors D and E), appears to disrupt functional dimers and target them for destruction by the proteasome. El apparently does not interact with microtubules directly and has no function in the tubulin folding pathway. Suppression of El expression seems to increase the cellular content of stable, posttranslationally modified microtubules by an unknown mechanism. Degradation of functional tubulin dimers as well as the alteration of the cellular content of stable microtubules through El might regulate the distribution and organization of organelles in vivo.     

Three-dimensional structure of human tubulin chaperone cofactor A

alpha and beta-Tubulin fold in a series of chaperone-assisted steps. At least five protein cofactors are involved in the post-chaperonin tubulin folding pathway and required to maintain the supply of tubulin; some of them also participate in microtubule dynamics. The first tubulin chaperone identified in the tubulin folding pathway was cofactor A (CoA). Here we describe the three-dimensional structure of human CoA at 1.7 A resolution, determined by multiwavelength anomalous diffraction (MAD). The structure is a monomer with a rod-like shape and consists of a three-alpha-helix bundle, or coiled coil, with the second helix kinked by a proline break, offering a convex surface at one face of the protein. The helices are connected by short turns, one of them, between alpha2 and alpha3, including a 3(10)-helix. Peptide mapping analysis and competition experiments with peptides show that CoA interacts with beta-tubulin via the three alpha-helical regions but not with the rod-end loops. The main interaction occurs with the middle kinked alpha2 helix, at the convex face of the rod. Strong 3D structural homology is found with the Hsp70 chaperone cofactor BAG domain, suggesting that these proteins define a family of cofactors of simple compact architecture. Further structural homology is found with alpha-spectrin/alpha-actinin repeats, all are rods of identical length of ten helical turns. We propose to call these three-helix bundles alpha ten modules.     

ADP ribosylation factor-like protein 2 (Arl2) regulates the interaction of tubulin-folding cofactor D with native tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the alpha/beta- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a beta-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.     

Role of cofactors B (TBCB) and E (TBCE) in tubulin heterodimer dissociation

Tubulin folding cofactors B (TBCB) and E (TBCE) are alpha-tubulin binding proteins that, together with Arl2 and cofactors D (TBCD), A (TBCA or p14) and C (TBCC), participate in tubulin biogenesis. TBCD and TBCE have also been implicated in microtubule dynamics through regulation of tubulin heterodimer dissociation. Understanding the in vivo function of these proteins will shed light on the Kenny-Caffey/Sanjad-Sakati syndrome, an important human disorder associated with TBCE. Here we show that, when overexpressed, TBCB depolymerizes microtubules. We found that this function is based on the ability of TBCB to form a binary complex with TBCE that greatly enhances the efficiency of this cofactor to dissociate tubulin in vivo and in vitro. We also show that TBCE, TBCB and alpha-tubulin form a ternary complex after heterodimer dissociation, whereas the free beta-tubulin subunit is recovered by TBCA. These complexes might serve to escort alpha-tubulin towards degradation or recycling, depending on the cell requirements.     

Gly-103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 protein is critical for DNA binding

Rad51 is a homolog of the bacterial RecA protein and is central for recombination in eukaryotes performing homology search and DNA strand exchange. Rad51 and RecA share a core ATPase domain that is structurally similar to the ATPase domains of helicases and the F1 ATPase. Rad51 has an additional N-terminal domain, whereas RecA protein has an additional C-terminal domain. Here we show that glycine 103 in the N-terminal domain of Saccharomyces cerevisiae Rad51 is important for binding to single-stranded and duplex DNA. The Rad51-G103E mutant protein is deficient in DNA strand exchange and ATPase activity due to a primary DNA binding defect. The N-terminal domain of Rad51 is connected to the ATPase core through an extended elbow linker that ensures flexibility of the N-terminal domain. Molecular modeling of the Rad51-G103E mutant protein shows that the negatively charged glutamate residue lies on the surface of the N-terminal domain facing a positively charged patch composed of Arg-260, His-302, and Lys-305 on the ATPase core domain. A possible structural explanation for the DNA binding defect is that a charge interaction between Glu-103 and the positive patch restricts the flexibility of the N-terminal domain. Rad51-G103E was identified in a screen for Rad51 interaction-deficient mutants and was shown to ablate the Rad54 interaction in two-hybrid assays (Krejci, L., Damborsky, J., Thomsen, B., Duno, M., and Bendixen, C. (2001) Mol. Cell. Biol. 21, 966-976). Surprisingly, we found that the physical interaction of Rad51-G103E with Rad54 was not affected. Our data suggest that the two-hybrid interaction defect was an indirect consequence of the DNA binding defect.     

Crystal structure of the post-chaperonin beta-tubulin binding cofactor Rbl2p.

The folding pathway of tubulins includes highly specific interactions with a series of cofactors (A, B, C, D and E) after they are released from the eukaryotic chaperonin CCT. The 2.2 A crystal structure of Rbl2p, the Saccharomyces cerevisiae homolog of beta-tubulin specific cofactor A, shows alpha-helical monomers forming a flat, slightly convex dimer. The surface of the molecule is dominated by polar and charged residues and lacks hydrophobic patches typically observed for chaperones that bind unfolded or partially folded proteins. This post-chaperonin cofactor is therefore clearly distinct from typical chaperones where hydrophobicity is a hallmark of substrate recognition.     

Cofactor D functions as a centrosomal protein and is required for the recruitment of the gamma-tubulin ring complex at centrosomes and organization of the mitotic spindle

Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.     

Characterisation of fission yeast alp11 mutants defines three functional domains within tubulin-folding cofactor B

The proper folding of tubulins prior to their incorporation into microtubules requires a group of conserved proteins called cofactors A to E. In fission yeast, homologues of these cofactors (at least B, D and E) are necessary for the biogenesis of microtubules and for cell viability. Here we show that the temperature-sensitive alp11-924 mutant, which is defective in the cofactor B homologue, contains an opal nonsense mutation, which results in the production of a truncated Alp11^B protein (Alp11_1–118). We isolated a tRNA^Trp gene as a multicopy suppressor of this mutation, which rescues alp11-924 by read-through of the nonsense codon. The truncated Alp11_1–118 protein lacks the C-terminal half of Alp11^B, consisting of a central coiled-coil region and the distal CLIP-170 domain found in a number of proteins involved in microtubule functions. Both of these domains are required for the maintenance of microtubule architecture in vivo. Detailed functional analyses lead us to propose that Alp11^B comprises three functional domains: the N-terminal half executes the essential function, the central coiled-coil region is necessary for satisfactory maintenance of cellular α-tubulin levels, and the C-terminal CLIP-170 domain is required for efficient binding to α-tubulin. Received: 29 November 1999 / Accepted: 18 April 2000     

UBXD1 binds p97 through two independent binding sites

The chaperone-related p97 protein plays a central role in various cellular processes involving the ubiquitin-proteasome system. The diverse functions of p97 are controlled by a large number of cofactors that recruit specific substrates or influence their ubiquitylation state. Many cofactors bind through a UBX or PUB domain, two major p97 binding modules. However, the recently identified UBXD1 cofactor possesses both domains. To elucidate the molecular basis underlying the interaction between UBXD1 and p97, we analyzed the contribution of both domains to p97 binding biochemically and in living cells. The PUB domain mediated robust binding to the carboxy-terminus of p97, while the UBX domain did not contribute to p97 binding. Importantly, we identified an additional p97 binding site in UBXD1 that competed with the p47 cofactor for binding to the N domain of p97. This novel, bipartite binding mode suggests that UBXD1 could be an efficient regulator of p97 cofactor interactions.     

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding

Villin-type headpiece domains are ～70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.     

The alpha- and beta-tubulin folding pathways

The alpha-beta tubulin heterodimer is the subunit from which microtubules are assembled. The pathway leading to correctly folded alpha- and beta-tubulins is unusually complex: it involves cycles of ATP-dependent interaction of newly synthesized tubulin subunits with cytosolic chaperonin, resulting in the production of quasi-native folding intermediates, which must then be acted upon by additional protein cofactors. These cofactors form a supercomplex containing both alpha- and beta-tubulin polypeptides, from which native heterodimer is released in a GTP-dependent reaction. Here, we discuss the current state of our understanding of the function of cytosolic chaperonin and cofactors in tubulin folding.     

Review: postchaperonin tubulin folding cofactors and their role in microtubule dynamics

The microtubule cytoskeleton consists of a highly organized network of microtubule polymers bound to their accessory proteins: microtubule-associated proteins, molecular motors, and microtubule-organizing proteins. The microtubule subunits are heterodimers composed of one alpha-tubulin polypeptide and one beta-tubulin polypeptide that should undergo a complex folding processing before they achieve a quaternary structure that will allow their incorporation into the polymer. Due to the extremely high protein concentration that exists at the cell cytoplasm, there are alpha- and beta-tubulin interacting proteins that prevent the unwanted interaction of these polypeptides with the surrounding protein pool during folding, thus allowing microtubule dynamics. Several years ago, the development of a nondenaturing electrophoretic technique made it possible to identify different tubulin intermediate complexes during tubulin biogenesis in vitro. By these means, the cytosolic chaperonin containing TCP-1 (CCT or TriC) and prefoldin have been demonstrated to intervene through tubulin and actin folding. Various other cofactors also identified along the alpha- and beta-tubulin postchaperonin folding route are now known to have additional roles in tubulin biogenesis such as participating in the synthesis, transport, and storage of alpha- and beta-tubulin. The future characterization of the tubulin-binding sites to these proteins, and perhaps other still unknown proteins, will help in the development of chemicals that could interfere with tubulin folding and thus modulating microtubule dynamics. In this paper, current knowledge of the above postchaperonin folding cofactors, which are in fact chaperones involved in tubulin heterodimer quaternary structure achievement, will be reviewed.     

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.     

Crystal Structures of Bacillus subtilis Lon Protease

Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.     

The general definition of the p97/valosin-containing protein (VCP)-interacting motif (VIM) delineates a new family of p97 cofactors

Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatory X (UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX(5)AAX(2)R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors.     

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.     

Sequence-structure-function analysis of the bifunctional enzyme MnmC that catalyses the last two steps in the biosynthesis of hypermodified nucleoside mnm5s2U in tRNA

MnmC catalyses the last two steps in the biosynthesis of 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U) in tRNA. Previously, we reported that this bifunctional enzyme is encoded by the yfcK open reading frame in the Escherichia coli K12 genome. However, the mechanism of its activity, in particular the potential structural and functional dependence of the domains responsible for catalyzing the two modification reactions, remains unknown. With the aid of the protein fold-recognition method, we constructed a structural model of MnmC in complex with the ligands and target nucleosides and studied the role of individual amino acids and entire domains by site-directed and deletion mutagenesis, respectively. We found out that the N-terminal domain contains residues responsible for binding of the S-adenosylmethionine cofactor and catalyzing the methylation of nm(5)s(2)U to form mnm(5)s(2)U, while the C-terminal domain contains residues responsible for binding of the FAD cofactor. Further, point mutants with compromised activity of either domain can complement each other to restore a fully functional enzyme. Thus, in the conserved fusion protein MnmC, the individual domains retain independence as enzymes. Interestingly, the N-terminal domain is capable of independent folding, while the isolated C-terminal domain is incapable of folding on its own, a situation similar to the one reported recently for the rRNA modification enzyme RsmC.