Tubulin code eraser CCP5 binds branch glutamates by substrate deformation

Tubulin code eraser CCP5 binds branch glutamates by substrate deformation

Post-translational modifications of tubulin such as acetylation, detyrosination, and glutamylation profoundly affect tubulin’s structure and function. Glutamylation of tubulin, which is the addition of glutamate branches, is a widespread event on tubulin c-terminal tails and is regulated through the balance of tubulin tyrosine ligase like (TTLL) and cytosolic carboxypeptidases (CCP) enzymes.  While the metallocarboxypeptide CCP proteins are known to deglutamylate tubulins, the precise mechanisms by which they recognize and interact with tubulin are still under intense investigation.  Work by the Roll-Mecak lab recently shed light on how CCP5 specifically interacts with tubulin to regulate its glutamylation state.  Preliminary studies sought to understand the specific substrate for CCP5 as there were conflicting reports in the literature.  The group utilized purified CCP5 in combination with synthesized glutamylated alpha and beta tubulin tail peptides to show that CCP5 substrate is branched, mono-glutamylated tubulin tails; these findings were confirmed with TIRF microscopy assays.  X-ray crystallography of CCP5 alone (residues 2-605 delta loop) and cryo-EM structure of CCP5 (2-605) in complex with microtubules were then performed to define the structural basis for substrate recognition.  In-depth analysis and structural refinements showed that the CCP5 enzyme is conformationally flexible on the tubulin, but several critical residues and structural regions were identified.  A synthetic inhibitor was developed to provide additional insight into how the CCP5 active sight interacts with branched glutamates, and it was shown that there was a specific interaction akin to a lock-and-key.  Conversely, the group showed that CCP5 has broad recognition for tubulin backbone binding, and was performed by the N- and C-domains of CCP5.  Interestingly, CCP5 induces a unique turn in the tubulin tail which was necessary for glutamylation recognition.  Cytoskeleton Inc.’s Tubulin products  (Cat. # T238P and Cat. # T333P) were critical reagents used in these structural studies that provided insight into glutamate branch recognition by CCP5.