Cryo-EM structure of VASH1-SVBP bound to microtubules

Abstract

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.

Comprehensive Analysis of Binding Sites in Tubulin

“…We found a few cases where protein partners did not bind to one of the computationally predicted tubulin pockets or crystallographic fragment sites. These are the motor domains of motile kinesins, [30] the CH domains of end binding proteins (EBs), [19, 31] the CKK domain of the microtubule minus‐end‐targeting proteins CAMSAPs, [32] the spectrin domain of the protein regulator of cytokinesis 1 (PRC1), [33] the second and third helical motifs of the chlamydial type three secretion effector protein CopN, [29] the vasohibin‐SVBP complex, [34] and two synthetic protein binders [27, 29] . With the exception of latter, all these interactions are mediated through large shallow, composite binding sites formed either at the intra‐tubulin dimer interface or at inter‐tubulin dimer interfaces formed between two or four tubulin dimers in the microtubule lattice.…”

Molecular determinants for α-tubulin methylation by SETD2

“…For example, tubulin tyrosine ligase (TTL) makes critical electrostatic interactions with α-tubulin residues E445, E446, and E447 in order to align the CTT within the active site (52). The VASH-SVBP complex that removes the terminal tyrosine from α-tubulin, clear density of the CTT remains unresolved by both x-ray crystallography and cryo electron microscopy (53, 54) makes electrostatic interactions with a compound, epoY, that mimics the tubulin CTT residues (55). Similarly, the tubulin CTTs are essential for microtubule recognition by tubulin tyrosine ligase-like 7 (TTLL), an enzyme that adds glutamate chains to the CTTs of both α- and β-tubulin (56, 57).…”

The Emerging Roles of Axonemal Glutamylation in Regulation of Cilia Architecture and Functions

“…However, owing to the non-specific enzymatic activities and poor spatiotemporal accuracy of PTM perturbation with the current methods, caution is needed with respect to the interpretation of the roles of PTMs in cellular functions. Increasing structural and biochemical evidence has shown the detailed mechanisms by which tubulin PTM–modifying enzymes interact with microtubules and execute their enzymatic reactions ( Mukai et al, 2009 ; Otero et al, 2012 ; Tort et al, 2014 ; Garnham et al, 2015 , 2017 ; Wu et al, 2015 ; Miyake et al, 2016 ; Park et al, 2016 ; Natarajan et al, 2017 ; Skultetyova et al, 2017 ; Adamopoulos et al, 2019 ; Eshun-Wilson et al, 2019 ; Li et al, 2019 , 2020 ; Liao et al, 2019 ; Liu et al, 2019 ; Wang N. et al, 2019 ; Zhou et al, 2019 ; Janke and Magiera, 2020 ; Mahalingan et al, 2020 ; Ustinova et al, 2020 ). These studies provide important fundamental information for engineering tubulin PTM–modifying enzymes with better substrate specificities and vigorous enzyme activity.…”