Crystal structure of the tubulin tyrosine carboxypeptidase complex VASH1–SVBP

Abstract

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.Microtubules are key components of the eukaryotic cytoskeleton, involved in cell division, morphogenesis, motility and intracellular transport. Post-translational modifications of tubulin heterodimers, the so-called 'tubulin code' , includes the enzymatic removal and ligation of the C-terminal tyrosine 1 . Although the tubulin tyrosine ligase that reverts α-tubulin to the translated form has been described 2 and structurally characterized 3,4 , the carboxypeptidase that removes it has remained elusive for four decades. Recently, we 5 and others 6 described a complex between SVBP, a 66-residue peptide, and vasohibins (VASH1 or VASH2), as the long-sought-for tubulin carboxypeptidases. Vasohibins increase detyrosination of α-tubulin in cells and in vitro 5 , especially in the presence of SVBP, and both proteins have been implicated in neuronal function, a role that may be associated with their role in tubulin detyrosination 6 .A chaperone-like function was possible because SVBP enhances the levels of detyrosinated α-tubulin, and concomitantly affects the cellular abundance and solubility of vasohibins. VASH1 might have a transglutaminase-like protease fold, with a non-canonical Cys-His-Ser catalytic triad 7 ; however, low similarity to existing structures precludes establishment of a reliable structural model. To study the folding of VASH1, to understand how SVBP affects vasohibins and to examine how VASH1 recognizes and cleaves the α-tubulin C-terminal tyrosine, we co-expressed a VASH1-SVBP complex in insect cells, and then purified and crystallized it. The crystal structure was determined by sulfur single-wavelength anomalous dispersion (S-SAD) phasing, notably averaging 16 data sets of 360° sweeps using a PRIGo multi-axis goniometer 8 . The structure was refined to 2.1 Å resolution to an R free of 21.4% (see Methods and Supplementary Table 1 for crystallographic details).Only residues 60-304 of VASH1 (1-315 expressed) and 26-52 of SVBP (1-66) were visible in the electron density maps and modeled

Evolutionary Divergence of Enzymatic Mechanisms for Tubulin Detyrosination

“…Altogether, these data indicate that binding occurs through the corresponding region of VASH2 (N-terminal residues 66-104) and VASH1 (N-terminal 76-115), a domain we termed the SVBPbinding site (SVBP-BS). The very recent structural data obtained for VASH1 and VASH2 (Adamopoulos et al, 2019;Liao et al, 2019) allowed structural alignment of the SVBP-BS region (Figure 1E) and confirmed the presence of important residues within this region that are involved in interaction with SVBP. Remarkably, co-transfection of either VASH1 or VASH2 with SVBP resulted in a marked increase in total tubulin detyrosination compared with cells transfected with the enzymes alone ( Figures 2A and 2B).…”

“…. Surprisingly, changing the COOH group of tyrosine to NH 2 completely abolished the inhibitory effect of the compounds ( Figure 7C, compare lanes 3 and 4), likely because of the loss of a stabilizing salt bridge between the inhibitor and the arginine surrounding the active pocket (Adamopoulos et al, 2019;Liao et al, 2019). Moreover, the tripeptide EQY alone or modified with a reactive NO 2 group (compound 3) did not result in any detectable TbVASH inhibition (Figure 7C, compare lanes 3, 5, and 6).…”

Cryo-EM structure of VASH1-SVBP bound to microtubules

“…How vasohibins recognize the C-terminal tail of α-tubulin (CTα) is unresolved. Based on crystal structures of VASH1/2 bound to substrate-mimicking covalent inhibitors and subsequent mutagenesis data, we and two other groups identified the positively charged groove near the active site as the CTα-binding site ( Adamopoulos et al, 2019 ; Li et al, 2019 ; Wang et al, 2019 ). A later study challenged this model, however, and showed that the free CTα peptide did not bind to the positively charged groove of VASH2, but instead bound to a site on the opposite side of the active site, with the backbone of CTα orientated in the opposite direction ( Zhou et al, 2019 ).…”

“…In contrast, VASH1/2-SVBP preferably detyrosinate polymerized microtubules ( Li et al, 2019 ; Nieuwenhuis et al, 2017 ). The crystal structures of VASH1/2-SVBP have revealed how these enzymes recognize the C-terminal tyrosine ( Adamopoulos et al, 2019 ; Li et al, 2019 ; Liao et al, 2019 ; Wang et al, 2019 ; Zhou et al, 2019 ), but the structural basis for their substrate specificity towards microtubules is not understood. In particular, it remains to be determined whether VASH1/2-SVBP make additional contacts with the microtubule lattice and whether they can distinguish the α-tubulin conformations in free αβ-tubulin heterodimers and polymerized microtubules.…”

The crystal structure of the tetrameric human vasohibin-1–SVBP complex reveals a variable arm region within the structural core

“…Sequence analysis of vasohibins identified that they belong to the transglutaminase-like cysteine proteases, with conserved catalytic residues and a putative Ca 2+ -binding site (Sanchez-Pulido & Ponting, 2016). Crystal structures of the vasohibin-SVBP complex revealed that the catalytic site is indeed similar to that of transglutaminase (Adamopoulos et al, 2019;Li et al, 2019;Liao et al, 2019;Wang et al, 2019;Zhou et al, 2019;Liu et al, 2019). SVBP binds to the N-terminal structural core region of vasohibin and stabilizes the complex.…”