Molecular Determinants of Tubulin’s C-Terminal Tail Conformational Ensemble

Wall, Kathryn P.; Pagratis, Maria; Armstrong, Geoffrey S.; Balsbaugh, Jeremy L.; Verbeke, Eric J.; Pearson, Chad G.; Hough, Loren E.

doi:10.1021/acschembio.6b00507

Cited by 39 publications

(57 citation statements)

References 61 publications

(169 reference statements)

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“…Tubulin was purified from wild-type T. thermophila grown in minimal bacterized media as previously described for isotopic labeling (34). GST-CTT peptides were expressed and purified as previously described (34). T 1 and T 2 relaxation experiments were performed using the standard 15 N-HSQC experiment from the Varian BioPac (gNhsqc) on an 800 MHz magnet.…”

Section: N Relaxation Nmr (R 1 /R 2 )mentioning

confidence: 99%

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C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

Wall

Hart

Lee

et al. 2019

Preprint

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Microtubules are biopolymers that perform diverse cellular functions. The regulation of microtubule behavior occurs in part through post-translational modification of both the ↵and -subunits of tubulin. One class of modifications is the heterogeneous addition of glycine and glutamate residues to the disordered C-terminal tails of tubulin. Due to their prevalence in stable, high stress cellular structures such as cilia, we sought to determine if these modifications alter the intrinsic sti ness of microtubules. Here we describe the purification and characterization of di erentially-modified pools of tubulin from Tetrahymena thermophila. We found that glycylation on the ↵-C-terminal tail is a key determinant of microtubule sti ness, but does not a ect the number of protofilaments incorporated into microtubules. We measured the dynamics of the tail peptide backbone using nuclear magnetic resonance spectroscopy. We found that the spin-spin relaxation rate (R 2 ) showed a pronounced decreased as a function of distance from the tubulin surface for the ↵-tubulin tail, indicating that the ↵-tubulin tail interacts with the dimer surface. This suggests that the interactions of the ↵-C-terminal tail with the tubulin body contributes to the sti ness of the assembled microtubule, providing insight into the mechanism by which glycylation and glutamylation can alter microtubule mechanical properties.SIGNIFICANCE Microtubules are regulated in part by post-translational modifications including the heterogeneous addition of glycine and glutamate residues to the C-terminal tails. By producing and characterizing di erentially-modified tubulin, this work provides insight into the molecular mechanisms of how these modifications alter intrinsic microtubule properties such as flexibility. These results have broader implications for mechanisms of how ciliary structures are able to function under high stress.

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Section: N Relaxation Nmr (R 1 /R 2 )mentioning

confidence: 99%

“…Our NMR spectrum contains more than one resonance for . Several residues in our NMR spectra are present in two di erent chemical environments (34). For residues near the C-termini, these correspond to modification state (orange: polyglycylation of the i-1 residue, green: mono-glycylation of the i-1 residue).…”

Section: ↵-Tubulin Tail With the Tubulin Bodymentioning

confidence: 99%

C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

Wall

Hart

Lee

et al. 2019

Preprint

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“…As we have demonstrated recently in the studies of U- 13 C, 15 N-CAP-Gly domain of mammalian dynactin assembled with polymeric microtubules, the first method reports on both the direct interactions and allosteric structural perturbations while the second one is not applicable to CAP-Gly/microtubule assemblies as isotopic labeling of microtubules cannot be readily performed at this time (Wall et al 2016). We have therefore explored an alternative approach for gaining atomic-resolution information into the interfaces formed by U- 13 C, 15 N-CAP-Gly and natural abundance microtubules, dubbed dREDOR.…”

Section: Introductionmentioning

confidence: 99%

Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

Guo

Hou

et al. 2017

J Biomol NMR

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REDOR-based experiments with simultaneous 1H−13C and 1H−15N dipolar dephasing are explored for investigating intermolecular protein-protein interfaces in complexes formed by a U-13C,15N-labeled protein and its natural abundance binding partner. The application of a double-REDOR filter (dREDOR) results in a complete dephasing of proton magnetization in the U-13C,15N-enriched molecule while the proton magnetization of the unlabeled binding partner is not dephased. This retained proton magnetization is then transferred across the intermolecular interface by 1H-13C or 1H-15N cross polarization, permitting to establish the residues of the U-13C,15N-labeled protein, which constitute the binding interface. To assign the interface residues, this dREDOR-CPMAS element is incorporated as a building block into 13C-13C correlation experiments. We established the validity of this approach on U-13C,15N-Histidine and on a structurally characterized complex of dynactin’s U-13C,15N-CAP-Gly domain with end-binding protein 1 (EB1). The approach introduced here is broadly applicable to the analysis of intermolecular interfaces when one of the binding partners in a complex cannot be isotopically labeled.

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“…Tubulin glycylation is conserved in protists and most metazoans (17). The glycyl chain length on either α-or β-tubulin tails in axonemes is highly variable (16)(17)(18), ranging from 1 to 40 posttranslationally added glycines (19). Of the 13 TTLL enzymes in mammals (10, 11), three are glycylases: TTLL3, -8, and -10 (12,13,20).…”

mentioning

confidence: 99%

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

Garnham

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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.tubulin code | TTLL enzymes | glycylation | tubulin modification | glutamylation

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Molecular Determinants of Tubulin’s C-Terminal Tail Conformational Ensemble

Cited by 39 publications

References 61 publications

C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

C-terminal tail polyglycylation and polyglutamylation alter microtubule mechanical properties

Mapping protein–protein interactions by double-REDOR-filtered magic angle spinning NMR spectroscopy

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

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