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.

Highlights d The crystal structure of human Spartan bound to DNA is determined d The SprT domain contains a Zn 2+-binding subdomain that binds single-stranded DNA d DNA engages two Spartan molecules with a conserved dimer interface in the crystal d DNA positions the SprT domain for optimal cleavage of DNAprotein crosslinks

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.