LAUSR

Dear Editor, Microtubules are major elements of the cytoskeleton in eukaryotic cells, essential to a wide variety of cellular functions, including cell shape control, cell division, morphogenesis, motility, and motor protein-based intracellular transport. Microtubules are dynamic cylindrical polymers assembled from the conserved αand β-tubulin heterodimers, which contain globular cores forming the tubular structure and negatively charged C-terminal tails with more variable forms exposed at the microtubule surface. Despite the conservation, cells use multiple αand β-tubulin isoforms with chemically diverse post-translational modifications (PTMs) to adapt to the specialized functions of microtubules. These PTMs include detyrosination/tyrosination, acetylation/deacetylation, polyglutamylation, and polyglycylation, etc. Various tubulin isoforms and abundant PTMs are collectively known as the “tubulin code”, which regulates microtubule properties and its interaction with microtubule-associated protein (MAPs). Detyrosination/tyrosination is one PTM that involves cyclic removal and reincorporation of the C-terminal tyrosine on most α-tubulin isotypes. Tubulin tyrosine ligase (TTL) catalyzes the retyrosination of detyrosinated α-tubulin, while recently vasohibins (VASHs)/SVBP complex were reported as the long-sought tubulindetyrosinating enzymes, encoding tubulin carboxypeptidase (TCP) activity. Two VASHs (VASH1 and VASH2) are found in mammalian genomes, with ~50% sequence identity. VASHs were first identified as secreted angiogenesis regulators, and predicted to harbor a transglutaminase-like protease fold. Small vasohibin binding protein (SVBP), a 66-residue protein, acts as a chaperone-like peptide, which is helpful for vasohibin stability and enhances the tubulin carboxypeptidase activity. The identification of VASHs as major tubulindetyrosinating enzymes establishes the nature of molecules that start the detyrosination-tyrosination cycle, and this helps to extend the understanding of the tubulin PTM in cells. Despite genetic and biochemical studies into the tubulin carboxypeptidase activities of vasohibins/ SVBP, the detailed molecular mechanism underlying αtubulin detyrosination is still unclear due to the lack of structures of vasohibins/SVBP. In this study, we determined the crystal structure of human VASH1-SVBP complex, thereby providing insights into the molecular mechanism of α-tubulin detyrosination by the VASH1SVBP complex. Due to the poor solubility of VASH1 when expressed alone, we co-expressed the human SVBP with a C-terminal His tag and VASH1 with a C-terminal Strep tag. After purification tests with different fragments of VASH1, a catalytic core-containing complex VASH1-SVBP was purified to homogeneity and we solved the structure of the heterodimer at 2.28 Å resolution by Pt-SAD phasing (Fig. 1a, X-ray statistics in Supplementary Table S1). Importantly, the heterodimer was fully active in the detyrosination activity assays with the tubulin heterodimer or GST (glutathione S-transferase) fusion proteins with C-terminal extensions corresponding to the Cterminal sequence of TUBA1A (VEGEGEEEGEEY) as substrates (Fig. 1b, c). One VASH1-SVBP complex exists in the asymmetric unit. The final refined heterodimer structure contains residues from 60 to 304 of VASH1 and residues 25 to 53 of SVBP. The rest residues of the two proteins are invisible probably due to their intrinsic flexibility.