VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules

Abstract

The detyrosination/tyrosination cycle of alpha tubulin is critical for proper cell functioning. VASH1-SVBP and VASH2-SVBP are ubiquitous enzyme complexes involved in microtubule detyrosination. However, little is known about their mode of action. Here, we show in reconstituted systems and in cells that VASH1-SVBP and VASH2-SVBP drive global and local detyrosination of microtubules, respectively. We solved the cryo-electron microscopy structure of human VASH2-SVBP bound to microtubules, revealing a different microtubule-binding configuration of its central catalytic region compared to VASH1-SVBP. We further show that the divergent mode of detyrosination between the two enzymes is correlated with the microtubule-binding properties of their disordered N- and C-terminal regions. Specifically, the N-terminal region is responsible for a significantly longer residence time of VASH2-SVBP on microtubules compared to VASH1-SVBP. We suggest that this VASH domain is critical for microtubule-detachment and diffusion of VASH-SVBP enzymes on the lattice. Together, our results suggest a mechanism by which these enzymes could generate distinct microtubule subpopulations and confined areas of detyrosinated lattices to drive various microtubule-based cellular functions.

Detyrosination enrichment on microtubule subsets is established by the interplay between a stochastically-acting enzyme and microtubule stability

“…We have found no evidence suggesting that VASH changes its behavior if the α-tubulin tail is already detyrosinated, because we observed similar proportion of diffusive and confined motions when we tracked VASH early after nocodazole washout, when microtubules are mostly tyrosinated. These results are consistent with in vitro work showing very little differences in the interaction of VASH with tyrosinated and detyrosinated microtubules [45].…”

“…These results are consistent with previous literature showing conserved structures between these two isoforms. These results are also consistent with a recent in vitro study, which used total internal reflection fluorescence microscopy to track the binding of VASH1 and VASH2 to microtubules [45]. We note that in the in vitro work VASH2 residence time on the microtubule was dramatically longer than VASH1 residence time [45].…”

“…These results are also consistent with a recent in vitro study, which used total internal reflection fluorescence microscopy to track the binding of VASH1 and VASH2 to microtubules [45]. We note that in the in vitro work VASH2 residence time on the microtubule was dramatically longer than VASH1 residence time [45]. It is possible that in the cell context, presence of other microtubule associated proteins (MAPs) and PTMs dampens the dramatic differences in the binding mode of these two enzymes, as we found that the residence time in cells differ only slightly.…”

“…Mammals have two isoforms of VASH, VASH1 and VASH2, both of which constitutively complex with the small Vasohibin-binding protein (SVBP) [44] which is a 66-amino acid peptide containing mainly an alpha helix. The two VASH isoforms share conserved structures, including their catalytic cores, albeit recent Cryo-EM results showed some differences in their microtubule binding pose [32, 45].…”

“…For example, a recent study suggested that presence of MAP4, but not phosphorylated MAP4, on microtubules may block the local access of VASH2 in cardiomyocytes [63]. Previous cryo-EM studies showed that VASH, presumably stably bound to microtubules, engages with two α-tubulins from two adjacent protofilaments [32, 45], and structural studies also suggested that the C-terminal tail of α-tubulin is supposedly occupied by the catalytic pocket of VASH for detyrosination to occur [32, 33, 51, 52].…”