Tubulin code eraser CCP5 binds branch glutamates by substrate deformation.

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• Microtubule function relies on the tubulin code, a system of posttranslational modifications.
• Glutamylation, the addition of glutamate chains, is a widespread tubulin modification regulated by specific enzymes.
• Cytosolic carboxypeptidases (CCPs) are crucial for removing glutamate modifications, and their dysfunction is linked to human diseases.

Purpose of the Study:

• To elucidate the structural mechanisms by which CCP5 removes glutamate modifications from tubulin.
• To understand the molecular basis of glutamylation homeostasis and its role in cellular function.
• To investigate the substrate specificity and potential disease implications of CCP5 activity.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine the structure of CCP5 bound to microtubules.
• X-ray crystallography to visualize CCP5 in complex with transition-state analogues.
• Nuclear Magnetic Resonance (NMR) spectroscopy to analyze enzyme-substrate interactions.

Main Results:

• Determined high-resolution structures of CCP5 interacting with microtubules and transition-state analogues.
• Revealed that CCP5 induces a unique turn in the tubulin backbone for recognition of glutamate modifications.
• Identified a specific cationic pocket in CCP5 crucial for binding glutamate branches.
• Demonstrated CCP5's ability to process diverse tubulin isotypes and non-tubulin substrates via backbone interactions.
• Observed inefficient processing of a specific brain β-tubulin isotype by CCP5.

Conclusions:

• Provided an atomistic understanding of how CCP5 recognizes and removes glutamate modifications from tubulin.
• Highlighted the role of structural flexibility and specific binding pockets in CCP5's enzymatic activity.
• Suggested potential implications for tubulin glutamylation homeostasis and disease pathogenesis, particularly concerning specific β-tubulin isotypes in the brain.