Distinct Tubulin C-Terminal Tails Control the Efficiency of a Microtubule Severing Machine

Microtubule severing enzymes of the AAA+ family are essential regulators of cytoskeletal remodeling, extracting subunits from the microtubule lattice through ATP-driven conformational changes. Among them, katanin assembles into hexameric structures and binds the negatively charged carboxy-terminal tails (CTTs) of tubulin through its central pore. Experimental studies have shown that different tubulin CTT isotypes can act as either inhibitors or non-inhibitors of katanin-mediated severing, with increased CTT hydrophobicity associated with reduced inhibition. However, the molecular basis underlying this selective behavior remains poorly understood. Here, we employed molecular dynamics simulations, and quantitative analysis combining principal component analysis, clustering, and distance distribution analysis, to investigate how natural tubulin CTTs (beta5, beta4b, and beta3), and engineered CTTs (beta5-A+Y, beta5-cterm, beta5-midpoint, and poly-E) influence katanin structure and dynamics in spiral and ring conformations. Our results show that inhibitory CTTs form stronger interactions with the terminal protomers and increase flexibility in the inner protomers, resulting in coordinated motions associated with pore narrowing. In contrast, non-inhibitory CTTs preferentially interact with inner protomers, disrupt interprotomer coordination, weakening the collective grip of the hexamer on the substrate. Analyses of the engineered constructs further revealed that inhibitory behavior is governed primarily by the spatial distribution of acidic residues rather than the overall charge. Additionally, we identified species-specific responses of different CTT isotypes toward the katanin ring state. Together, these findings provide molecular-level insight into how tubulin CTT sequence organization regulates substrate recognition, pore dynamics, and severing efficiency, leading to predictive design of CTTs with a desired action on katanin.