Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility, and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes, and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.Microtubules are non-covalent cylindrical polymers formed by ␣␤-tubulin heterodimer building blocks. They possess two seemingly contradictory properties; they are highly dynamic, exhibiting rapid growth and shrinkage of their ends (1), but are also very rigid, with persistence lengths on the order of cellular dimensions (2). This duality is thought to underlie the versatile architectures of microtubule networks in cells ( Fig. 1) and is tuned by a myriad of cellular effectors. These fall into two categories: effectors that bind to the microtubule and alter its properties non-covalently (motors and microtubule-associated proteins (MAPs)) 2 and effectors that chemically modify the tubulin subunits (tubulin posttranslational modification enzymes). Although the field has made tremendous progress in recent decades identifying a compendium of microtubule-interacting proteins and understanding their interplay and regulation in the cell, we are just now starting to unravel the basic mechanisms used by cells to chemically modify microtubules, despite the fact that tubulin posttranslational modifications have been known for over 40 years. A renaissance of interest into the roles of tubulin posttranslational modifications has been precipitated by the discovery in the last few years of the enzymes responsible for these modifications (3-8), methods for producing unmodified (9, 10), engineered, (11, 12) as well as chemically defined modified tubulin (13), and developments and refinements of in vitro microtubule-based assays using high-resolution microscopy and microfabricated substrates (14 -16).Tubulin posttranslational modifications are chemically diverse, ranging from phosphorylation (17), acetylation (18,19), palmitoylation (20), sumoylation (21), polyamination (22), and S-nitrosylation (23) to tyrosination (24), glutamylation (25, 26), and glycylation (27). Most of these modifications are reversible. Tubulin posttranslational modifications are evolutionarily conserved and abundantly represented in cellular microtubules. Most importantly, their distribution is stereotyped in cells. For example, interphase microtubules are enriched in tyrosination (28), whereas kinetochore fibers a...

Glutamylation, the most prevalent tubulin posttranslational modification, marks stable microtubules and regulates recruitment and activity of microtubule- interacting proteins. Nine enzymes of the tubulin tyrosine ligase-like (TTLL) family catalyze glutamylation. TTLL7, the most abundant neuronal glutamylase, adds glutamates preferentially to the β–tubulin tail. Coupled with ensemble and single- molecule biochemistry, our hybrid X-ray and cryo-electron microscopy structure of TTLL7 bound to the microtubule delineates a tripartite microtubule recognition strategy. The enzyme uses its core to engage the disordered anionic tails of α– and β–tubulin, and a flexible cationic domain to bind the microtubule and position itself for β–tail modification. Furthermore, we demonstrate that all single-chain TTLLs with known glutamylase activity utilize a cationic microtubule-binding domain analogous to that of TTLL7. Therefore, our work reveals the combined use of folded and intrinsically disordered substrate recognition elements as the molecular basis for specificity among the enzymes primarily responsible for chemically diversifying cellular microtubules.

It is widely accepted that ubiquitin conjugating enzymes (E2) contain an active site asparagine that serves as an oxyanion hole, thereby stabilizing a negatively charged transition state intermediate and promoting ubiquitin transfer. Using structural and biochemical approaches to study the role of the conserved asparagine to ubiquitin conjugation by Ubc13/Mms2, we conclude that the importance of this residue stems primarily from its structural role in stabilizing an active site loop.