Multi-domain O-GlcNAcase structures reveal allosteric regulatory mechanisms

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Nucleocytoplasmic protein O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA).
• The O-GlcNAc hydrolase (OGA) enzyme possesses a pseudo-histone acetyltransferase (pHAT) domain, whose structure and function are largely unknown.
• Understanding OGA regulation is crucial for elucidating O-GlcNAc homeostasis.

Purpose of the Study:

• To determine the structure and function of the O-GlcNAc hydrolase (OGA) pseudo-histone acetyltransferase (pHAT) domain.
• To investigate the role of the pHAT domain in the multi-domain structure and activity of OGA.
• To reveal the allosteric mechanisms governing O-GlcNAc homeostasis.

Main Methods:

• X-ray crystallography to determine the structure of the Trichoplax adhaerens pHAT domain.
• Cryo-electron microscopy (cryo-EM) to resolve the structure of multi-domain T. adhaerens and human OGAs.
• Biophysical analyses to characterize OGA domain interactions and conformational flexibility.

Main Results:

• The eukaryotic OGA pHAT domain forms catalytically incompetent, symmetric homodimers with a putative peptide-binding site.
• In solution, OGA exists as flexible multi-domain dimers, with linker interactions restricting pHAT domain movement.
• pHAT domain movements in human OGA induce conformational changes in a flexible arm, remodeling the active site environment.

Conclusions:

• The OGA pHAT domain plays a crucial role in regulating O-GlcNAc hydrolase activity through allosteric mechanisms.
• Structural insights into the pHAT domain and its interactions provide a basis for understanding O-GlcNAc homeostasis.
• These findings uncover novel regulatory pathways impacting O-GlcNAc modification dynamics.