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Microtubules are regulated by posttranslational modifications (PTMs) of tubulin. The ligation and cleavage of the C-terminal tyrosine of α tubulin impact microtubule functions during mitosis, cardiomyocyte contraction, and neuronal processes. Tubulin tyrosination and detyrosination are mediated by tubulin tyrosine ligase (TTL) and the recently discovered tubulin detyrosinases, vasohibin 1 and 2 (VASH1 and VASH2) bound to the small vasohibin-binding protein (SVBP). Here, we report the crystal structures of human VASH1–SVBP alone, in complex with a tyrosine-derived covalent inhibitor, and bound to the natural product parthenolide. The structures and subsequent mutagenesis analyses explain the requirement for SVBP during tubulin detyrosination, and reveal the basis for the recognition of the C-terminal tyrosine and the acidic α tubulin tail by VASH1. The VASH1–SVBP–parthenolide structure provides a framework for designing more effective chemical inhibitors of vasohibins, which can be valuable for dissecting their biological functions and may have therapeutic potential.

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αβ-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αβ heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

The spindle checkpoint maintains genomic stability and prevents aneuploidy. Unattached kinetochores convert the latent open conformer of the checkpoint protein Mad2 (O-Mad2) to the active closed conformer (C-Mad2), bound to Cdc20. C-Mad2–Cdc20 is incorporated into the mitotic checkpoint complex (MCC), which inhibits the anaphase-promoting complex/cyclosome (APC/C). The C-Mad2-binding protein p31comet and the ATPase TRIP13 promote MCC disassembly and checkpoint silencing. Here, using nuclear magnetic resonance (NMR) spectroscopy, we show that TRIP13 and p31comet catalyze the conversion of C-Mad2 to O-Mad2, without disrupting its stably folded core. We determine the crystal structure of human TRIP13, and identify functional TRIP13 residues that mediate p31comet–Mad2 binding and couple ATP hydrolysis to local unfolding of Mad2. TRIP13 and p31comet prevent APC/C inhibition by MCC components, but cannot reactivate APC/C already bound to MCC. Therefore, TRIP13–p31comet intercepts and disassembles free MCC not bound to APC/C through mediating the local unfolding of the Mad2 C-terminal region., The spindle checkpoint ensures the fidelity of chromosome segregation during mitosis and meiosis. Here the authors use a combination of biochemical and structural biology approaches to show how the TRIP13 ATPase and its adaptor, p31comet, catalyze the conversion of the checkpoint protein Mad2 between latent and active forms

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients., Mutations in optineurin that cause defects in the interaction with TBK1 are associated with neurodegenerative diseases. Here, the authors report the structure of this complex, and outline a general binding mode for these proteins.

OPTN (optineurin), a ubiquitin-binding scaffold protein, functions as an important macroautophagy/autophagy receptor in selective autophagy processes. Mutations in OPTN have been linked with human neurodegenerative diseases including ALS and glaucoma. However, the mechanistic basis underlying the recognition of ubiquitin by OPTN and its regulation by TBK1-mediated phosphorylation are still elusive. Here, we demonstrate that the UBAN domain of OPTN preferentially recognizes linear ubiquitin chain and forms an asymmetric 2:1 stoichiometry complex with the linear diubiquitin. In addition, our results provide new mechanistic insights into how phosphorylation of UBAN would regulate the ubiquitin-binding ability of OPTN and how disease-associated mutations in the OPTN UBAN domain disrupt its interaction with ubiquitin. Finally, we show that defects in ubiquitin-binding may affect the recruitment of OPTN to linear ubiquitin-decorated mutant Huntington protein aggregates. Taken together, our findings clarify the interaction mode between UBAN and linear ubiquitin chain in general, and expand our knowledge of the molecular mechanism of ubiquitin-decorated substrates recognition by OPTN as well as the pathogenesis of neurodegenerative diseases caused by OPTN mutations.