Microtubules (MTs) undergo diverse posttranslational modifications that regulate their structural and functional properties. Among these, polyglutamylation-a dominant and conserved modification targeting unstructured tubulin C-terminal tails-plays a pivotal role in defining the tubulin code. Here, we describe a mechanism by which tubulin tyrosine ligase-like 11 (TTLL11) expands and diversifies the code. Cryo-electron microscopy revealed a unique bipartite MT recognition strategy wherein TTLL11 binding and catalytic domains engage adjacent MT protofilaments. Biochemical and cellular assays identified previously uncharacterized polyglutamylation patterns, showing that TTLL11 directly extends the primary polypeptide chains of α- and β-tubulin in vitro, challenging the prevailing paradigms emphasizing lateral branching. Moreover, cell-based and in vivo data suggest a cross-talk between polyglutamylation and the detyrosination/tyrosination cycle likely linked to the TTLL11-mediated elongation of the primary α-tubulin chain. These findings unveil an unrecognized layer of complexity within the tubulin code and offer mechanistic insights into the molecular basis of functional specialization of MT cytoskeleton.

Department of Biochemistry Faculty of Science Charles University Albertov 6 Prague Czech Republic

Department of Chemistry Umeå University SE 901 87 Umeå Sweden

Grenoble Institute Neurosciences University Grenoble Alpes Inserm U1216 CNRS 38000 Grenoble France

Institut de Biologie Structurale Université Grenoble Alpes CEA CNRS 38044 Grenoble France

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 Vestec Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 Prague Czech Republic

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