Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor

Area of Science:

• Structural biology
• Biochemistry
• Molecular mechanisms of protein function

Background:

• Bre5 protein functions as a cofactor for the deubiquitinating enzyme Ubp3.
• Bre5 possesses a nuclear transfer factor 2 (NTF2)-like protein recognition module crucial for Ubp3 activity.

Purpose of the Study:

• Determine the x-ray crystal structure of the Bre5 NTF2-like domain.
• Investigate the oligomeric state and binding interactions of Bre5 with Ubp3.
• Elucidate the molecular basis of Bre5-mediated regulation of Ubp3 function.

Main Methods:

• X-ray crystallography to determine protein structure.
• Sedimentation equilibrium studies to analyze protein oligomerization.
• Isothermal titration calorimetry to quantify binding affinities.
• Deletion analysis and site-directed mutagenesis to map interaction interfaces and functional domains.

Main Results:

• The Bre5 NTF2-like domain forms a homodimeric structure, stabilized by an intermolecular disulfide bond in crystals.
• Bre5 exists exclusively as a dimer in solution, while a mutant lacking the disulfide bond shows a midnanomolar monomer-dimer equilibrium.
• A novel N-terminal domain of Ubp3 mediates interaction with Bre5, forming a 2:1 Bre5-Ubp3 complex.
• The Ubp3 binding surface on Bre5 is located near the dimer interface and is essential for Ubp3 function.

Conclusions:

• Dimerization and disulfide bond formation of Bre5 may modulate its function as a Ubp3 cofactor.
• Bre5 interacts with Ubp3 in a unique 2:1 stoichiometry, distinct from other NTF2-like domain interactions.
• Structural insights into the Bre5-Ubp3 interaction provide a foundation for understanding Ubp3 regulation by its cofactor.