Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes1. Glutamylation-the addition of branched (isopeptide-linked) glutamate chains-is the most evolutionarily widespread tubulin modification2. It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family1. Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function3-9, and mutations in CCPs lead to human disease10-13. Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant ß-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax.

Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing.

Microtubules have spatiotemporally complex posttranslational modification patterns. How cells interpret this tubulin modification code is largely unknown. We show that C. elegans katanin, a microtubule severing AAA ATPase mutated in microcephaly and critical for cell division, axonal elongation, and cilia biogenesis, responds precisely, differentially, and combinatorially to three chemically distinct tubulin modifications-glycylation, glutamylation, and tyrosination-but is insensitive to acetylation. Glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing. Katanin exhibits graded and divergent responses to glutamylation on the α- and ß-tubulin tails, and these act combinatorially. The katanin hexamer central pore constrains the polyglutamate chain patterns on ß-tails recognized productively. Elements distal to the katanin AAA core sense α-tubulin tyrosination, and detyrosination downregulates severing. The multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes.

α-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics.

Microtubules are non-covalent polymers of αß-tubulin dimers. Posttranslational processing of the intrinsically disordered C-terminal α-tubulin tail produces detyrosinated and Δ2-tubulin. Although these are widely employed as proxies for stable cellular microtubules, their effect (and of the α-tail) on microtubule dynamics remains uncharacterized. Using recombinant, engineered human tubulins, we now find that neither detyrosinated nor Δ2-tubulin affect microtubule dynamics, while the α-tubulin tail is an inhibitor of microtubule growth. Consistent with the latter, molecular dynamics simulations show the α-tubulin tail transiently occluding the longitudinal microtubule polymerization interface. The marked differential in vivo stabilities of the modified microtubule subpopulations, therefore, must result exclusively from selective effector recruitment. We find that tyrosination quantitatively tunes CLIP-170 density at the growing plus end and that CLIP170 and EB1 synergize to selectively upregulate the dynamicity of tyrosinated microtubules. Modification-dependent recruitment of regulators thereby results in microtubule subpopulations with distinct dynamics, a tenet of the tubulin code hypothesis.

Author Correction: An allosteric network in spastin couples multiple activities required for microtubule severing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Katanin Grips the ß-Tubulin Tail through an Electropositive Double Spiral to Sever Microtubules.

The AAA ATPase katanin severs microtubules. It is critical in cell division, centriole biogenesis, and neuronal morphogenesis. Its mutation causes microcephaly. The microtubule templates katanin hexamerization and activates its ATPase. The structural basis for these activities and how they lead to severing is unknown. Here, we show that ß-tubulin tails are necessary and sufficient for severing. Cryoelectron microscopy (cryo-EM) structures reveal the essential tubulin tail glutamates gripped by a double spiral of electropositive loops lining the katanin central pore. Each spiral couples allosterically to the ATPase and binds alternating, successive substrate residues, with consecutive residues coordinated by adjacent protomers. This tightly couples tail binding, hexamerization, and ATPase activation. Hexamer structures in different states suggest an ATPase-driven, ratchet-like translocation of the tubulin tail through the pore. A disordered region outside the AAA core anchors katanin to the microtubule while the AAA motor exerts the forces that extract tubulin dimers and sever the microtubule.

An allosteric network in spastin couples multiple activities required for microtubule severing.

The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.

Katanin spiral and ring structures shed light on power stroke for microtubule severing.

Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

Structure and Dynamics of Single-isoform Recombinant Neuronal Human Tubulin.

Microtubules are polymers that cycle stochastically between polymerization and depolymerization, i.e. they exhibit "dynamic instability." This behavior is crucial for cell division, motility, and differentiation. Although studies in the last decade have made fundamental breakthroughs in our understanding of how cellular effectors modulate microtubule dynamics, analysis of the relationship between tubulin sequence, structure, and dynamics has been held back by a lack of dynamics measurements with and structural characterization of homogeneous isotypically pure engineered tubulin. Here, we report for the first time the cryo-EM structure and in vitro dynamics parameters of recombinant isotypically pure human tubulin. α1A/ßIII is a purely neuronal tubulin isoform. The 4.2-Å structure of post-translationally unmodified human α1A/ßIII microtubules shows overall similarity to that of heterogeneous brain microtubules, but it is distinguished by subtle differences at polymerization interfaces, which are hot spots for sequence divergence between tubulin isoforms. In vitro dynamics assays show that, like mosaic brain microtubules, recombinant homogeneous microtubules undergo dynamic instability, but they polymerize slower and have fewer catastrophes. Interestingly, we find that epitaxial growth of α1A/ßIII microtubules from heterogeneous brain seeds is inefficient but can be fully rescued by incorporating as little as 5% of brain tubulin into the homogeneous α1A/ßIII lattice. Our study establishes a system to examine the structure and dynamics of mammalian microtubules with well defined tubulin species and is a first and necessary step toward uncovering how tubulin genetic and chemical diversity is exploited to modulate intrinsic microtubule dynamics.

Multivalent Microtubule Recognition by Tubulin Tyrosine Ligase-like Family Glutamylases.

Glutamylation, the most prevalent tubulin posttranslational modification, marks stable microtubules and regulates recruitment and activity of microtubule- interacting proteins. Nine enzymes of the tubulin tyrosine ligase-like (TTLL) family catalyze glutamylation. TTLL7, the most abundant neuronal glutamylase, adds glutamates preferentially to the ß-tubulin tail. Coupled with ensemble and single-molecule biochemistry, our hybrid X-ray and cryo-electron microscopy structure of TTLL7 bound to the microtubule delineates a tripartite microtubule recognition strategy. The enzyme uses its core to engage the disordered anionic tails of α- and ß-tubulin, and a flexible cationic domain to bind the microtubule and position itself for ß-tail modification. Furthermore, we demonstrate that all single-chain TTLLs with known glutamylase activity utilize a cationic microtubule-binding domain analogous to that of TTLL7. Therefore, our work reveals the combined use of folded and intrinsically disordered substrate recognition elements as the molecular basis for specificity among the enzymes primarily responsible for chemically diversifying cellular microtubules.

Molecular basis for age-dependent microtubule acetylation by tubulin acetyltransferase.

Acetylation of α-tubulin Lys40 by tubulin acetyltransferase (TAT) is the only known posttranslational modification in the microtubule lumen. It marks stable microtubules and is required for polarity establishment and directional migration. Here, we elucidate the mechanistic underpinnings for TAT activity and its preference for microtubules with slow turnover. 1.35 Å TAT cocrystal structures with bisubstrate analogs constrain TAT action to the microtubule lumen and reveal Lys40 engaged in a suboptimal active site. Assays with diverse tubulin polymers show that TAT is stimulated by microtubule interprotofilament contacts. Unexpectedly, despite the confined intraluminal location of Lys40, TAT efficiently scans the microtubule bidirectionally and acetylates stochastically without preference for ends. First-principles modeling and single-molecule measurements demonstrate that TAT catalytic activity, not constrained luminal diffusion, is rate limiting for acetylation. Thus, because of its preference for microtubules over free tubulin and its modest catalytic rate, TAT can function as a slow clock for microtubule lifetimes.

Tubulin tyrosine ligase and stathmin compete for tubulin binding in vitro.

Tubulin partition between soluble and polymeric forms is tightly regulated in cells. Stathmin and tubulin tyrosine ligase (TTL) each form stable complexes with tubulin and inhibit tubulin polymerization. Here we explore the mutual relationship between these proteins in vitro and demonstrate that full-length stathmin and TTL compete for binding to tubulin and fail to make a stable tubulin:stathmin:TTL triple complex in solution. Moreover, stathmin depresses TTL tubulin tyrosination activity in vitro. These results suggest either that TTL and stathmin have a partially overlapping footprint on the tubulin dimer or that stathmin induces a tubulin conformation incompatible with stable TTL binding.

Crystal structures of tubulin acetyltransferase reveal a conserved catalytic core and the plasticity of the essential N terminus.

Tubulin acetyltransferase (TAT) acetylates Lys-40 of α-tubulin in the microtubule lumen. TAT is inefficient, and its activity is enhanced when tubulin is incorporated in microtubules. Acetylation is associated with stable microtubules and regulates the binding of microtubule motors and associated proteins. TAT is important in neuronal polarity and mechanosensation, and decreased tubulin acetylation levels are associated with axonal transport defects and neurodegeneration. We present the first structure of TAT in complex with acetyl-CoA (Ac-CoA) at 2.7 Å resolution. The structure reveals a conserved stable catalytic core shared with other GCN5 superfamily acetyltransferases consisting of a central ß-sheet flanked by α-helices and a C-terminal ß-hairpin unique to TAT. Structure-guided mutagenesis establishes the molecular determinants for Ac-CoA and tubulin substrate recognition. The wild-type TAT construct is a monomer in solution. We identify a metastable interface between the conserved core and N-terminal domain that modulates the oligomerization of TAT in solution and is essential for activity. The 2.45 Å resolution structure of an inactive TAT construct with an active site point mutation near this interface reveals a domain-swapped dimer in which the functionally essential N terminus shows evidence of marked structural plasticity. The sequence segment corresponding to this structurally plastic region in TAT has been implicated in substrate recognition in other GCN5 superfamily acetyltransferases. Our structures provide a rational platform for the mechanistic dissection of TAT activity and the design of TAT inhibitors with therapeutic potential in neuronal regeneration.

Tubulin tyrosine ligase structure reveals adaptation of an ancient fold to bind and modify tubulin.

Tubulin tyrosine ligase (TTL) catalyzes the post-translational C-terminal tyrosination of α-tubulin. Tyrosination regulates recruitment of microtubule-interacting proteins. TTL is essential. Its loss causes morphogenic abnormalities and is associated with cancers of poor prognosis. We present the first crystal structure of TTL (from Xenopus tropicalis), defining the structural scaffold upon which the diverse TTL-like family of tubulin-modifying enzymes is built. TTL recognizes tubulin using a bipartite strategy. It engages the tubulin tail through low-affinity, high-specificity interactions, and co-opts what is otherwise a homo-oligomerization interface in structurally related ATP grasp-fold enzymes to form a tight hetero-oligomeric complex with the tubulin body. Small-angle X-ray scattering and functional analyses reveal that TTL forms an elongated complex with the tubulin dimer and prevents its incorporation into microtubules by capping the tubulin longitudinal interface, possibly modulating the partition of tubulin between monomeric and polymeric forms.

Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis.

Nucleolar Essential Protein 1 (Nep1) is required for small subunit (SSU) ribosomal RNA (rRNA) maturation and is mutated in Bowen-Conradi Syndrome. Although yeast (Saccharomyces cerevisiae) Nep1 interacts with a consensus sequence found in three regions of SSU rRNA, the molecular details of the interaction are unknown. Nep1 is a SPOUT RNA methyltransferase, and can catalyze methylation at the N1 of pseudouridine. Nep1 is also involved in assembly of Rps19, an SSU ribosomal protein. Mutations in Nep1 that result in decreased methyl donor binding do not result in lethality, suggesting that enzymatic activity may not be required for function, and RNA binding may play a more important role. To study these interactions, the crystal structures of the scNep1 dimer and its complexes with RNA were determined. The results demonstrate that Nep1 recognizes its RNA site via base-specific interactions and stabilizes a stem-loop in the bound RNA. Furthermore, the RNA structure observed contradicts the predicted structures of the Nep1-binding sites within mature rRNA, suggesting that the Nep1 changes rRNA structure upon binding. Finally, a uridine base is bound in the active site of Nep1, positioned for a methyltransfer at the C5 position, supporting its role as an N1-specific pseudouridine methyltransferase.

Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole.

The ESX-1 secretion system plays a critical role in the virulence of M. tuberculosis and M. marinum, but the precise molecular and cellular mechanisms are not clearly defined. Virulent M. marinum is able to escape from the Mycobacterium-containing vacuole (MCV) into the host cell cytosol, polymerize actin, and spread from cell to cell. In this study, we have examined nine M. marinum ESX-1 mutants and the wild type by using fluorescence and electron microscopy detecting MCV membranes and actin polymerization. We conclude that ESX-1 plays an essential role in M. marinum escape from the MCV. We also show that the ESX-1 mutants acquire the ability to polymerize actin after being artificially delivered into the macrophage cytosol by hypotonic shock treatment, indicating that ESX-1 is not directly involved in initiation of actin polymerization. We provide evidence that M. marinum induces membrane pores approximately 4.5 nm in diameter, and this activity correlates with ESAT-6 secretion. Importantly, purified ESAT-6, but not the other ESX-1-secreted proteins, is able to cause dose-dependent pore formation in host cell membranes. These results suggest that ESAT-6 secreted by M. marinum ESX-1 could play a direct role in producing pores in MCV membranes, facilitating M. marinum escape from the vacuole and cell-to-cell spread. Our study provides new insight into the mechanism by which ESX-1 secretion and ESAT-6 enhance the virulence of mycobacterial infection.

Crystal structures of human alpha-defensins HNP4, HD5, and HD6.

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.

Crystal structure at 1.9A of E. coli ClpP with a peptide covalently bound at the active site.

ClpP, the proteolytic component of the ATP-dependent ClpAP and ClpXP chaperone/protease complexes, has 14 identical subunits organized in two stacked heptameric rings. The active sites are in an interior aqueous chamber accessible through axial channels. We have determined a 1.9 A crystal structure of Escherichia coli ClpP with benzyloxycarbonyl-leucyltyrosine chloromethyl ketone (Z-LY-CMK) bound at each active site. The complex mimics a tetrahedral intermediate during peptide cleavage, with the inhibitor covalently linked to the active site residues, Ser97 and His122. Binding is further stabilized by six hydrogen bonds between backbone atoms of the peptide and ClpP as well as by hydrophobic binding of the phenolic ring of tyrosine in the S1 pocket. The peptide portion of Z-LY-CMK displaces three water molecules in the native enzyme resulting in little change in the conformation of the peptide binding groove. The heptameric rings of ClpP-CMK are slightly more compact than in native ClpP, but overall structural changes were minimal (rmsd approximately 0.5 A). The side chain of Ser97 is rotated approximately 90 degrees in forming the covalent adduct with Z-LY-CMK, indicating that rearrangement of the active site residues to a active configuration occurs upon substrate binding. The N-terminal peptide of ClpP-CMK is stabilized in a beta-hairpin conformation with the proximal N-terminal residues lining the axial channel and the loop extending beyond the apical surface of the heptameric ring. The lack of major substrate-induced conformational changes suggests that changes in ClpP structure needed to facilitate substrate entry or product release must be limited to rigid body motions affecting subunit packing or contacts between ClpP rings.

The structure of the extracellular domain of triggering receptor expressed on myeloid cells like transcript-1 and evidence for a naturally occurring soluble fragment.

Triggering receptor expressed on myeloid cells like transcript-1 (TLT-1) is an abundant platelet-specific, type I transmembrane receptor. The extracellular fragment of TLT-1 consists of a single, immunoglobulin-like domain connected to the platelet cell membrane by a linker region called the stalk. Here we present evidence that a soluble fragment of the TLT-1 extracellular domain is found in serum of humans and mice and that an isoform of similar mass is released from platelets following activation with thrombin. We also report the crystal structure of the immunoglobulin domain of TLT-1 determined at the resolution of 1.19 A. The structure of TLT-1 is similar to other immunoglobulin-like variable domains, particularly those of triggering receptor expressed on myeloid cells-1 (TREM-1), the natural killer cell-activating receptor NKp44, and the polymeric immunoglobulin receptor. Particularly interesting is a 17-amino acid segment of TLT-1, homologous to a fragment of murine TREM-1, which, in turn, showed activity in blocking the TREM-1-mediated inflammatory responses in mice. Structural similarity to TREM-1 and polymeric immunoglobulin receptor, and evidence for a naturally occurring soluble fragment of the TLT-1 extracellular domain, suggest that this immunoglobulin-like domain autonomously plays an as yet unidentified, functional role.

Sequence-altered peptide adopts optimum conformation for modification-dependent binding of the yeast tRNAPhe anticodon domain.

Amino acid contributions to protein recognition of naturally modified RNAs are not understood. Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure. Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed. Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure. The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.

Structure of the scorpion toxin BmBKTtx1 solved from single wavelength anomalous scattering of sulfur.

This report describes the crystal structure of the K(+) channel-blocking toxin, BmBKTx1, isolated recently from the venom of the scorpion Buthus martensi Karsch. This is only the second structure of the short-chain K(+) channel-blocking toxin from scorpion solved by means of X-ray crystallography. Additionally, reductive dimethylation of folded BmBKTx1 employed to induce its crystallization and solution of the structure based on the anomalous signal from the sulfur atoms make this example quite unique. The monomer of BmBKTx1 is formed by 31 amino acid residues, including 6 cysteines connected in 3 disulfide bridges. Crystals of this toxin belong to the space group P2(1) with two molecules present in the asymmetric unit. The unit cell parameters are a = 21.40 A, b=39.70 A, c=29.37 A, and beta-94.13 grades. Based on the high-quality dataset (anomalous signal) collected to the resolution 1.72A using the conventional X-radiation generator (lambda Cu, K alpha = 1.5478 A), the positions of sulfur atoms contributed by 12 cysteine residues have been identified, and subsequent improvement of the experimental phases have allowed structure solution. The final model was refined to the crystallographic R-factor of 0.166. The methyl groups on several lysine residues could be easily modeled into the electron density.