Molecular Tweezers Block the Functional Pore of a Protein Machine

Area of Science:

• Biochemistry
• Molecular Biology
• Supramolecular Chemistry

Background:

• The enzyme p97 (a hexameric AAA-ATPase) is crucial for protein quality control and cell survival.
• p97 functions by threading substrate proteins through its central pore, a process fueled by ATP hydrolysis.
• Targeting protein pores is a promising strategy for therapeutic intervention.

Purpose of the Study:

• To design and characterize symmetric multivalent tweezers as novel inhibitors for protein pores.
• To investigate the inhibitory mechanism of these tweezers on the p97 enzyme.
• To establish multivalent tweezers as a versatile supramolecular strategy for targeting protein pores.

Main Methods:

• Rational drug design guided by molecular modeling.
• Synthesis and characterization of C3-symmetric multivalent tweezers.
• Biophysical methods (fluorescence-based assays) and biochemical assays (ATPase activity, protein unfolding).
• Site-directed mutagenesis to validate binding sites and mechanism.

Main Results:

• Developed and characterized symmetric multivalent tweezers capable of binding to the p97 pore entrance.
• Demonstrated that tweezers inhibit p97 ATPase activity and substrate unfolding by blocking the pore.
• Confirmed specific binding to p97 and validated the pore-blocking mechanism through mutagenesis.
• Showcased C3-symmetric tweezers as potent inhibitors, consistent with p97's symmetry.

Conclusions:

• Multivalent tweezers represent a novel class of supramolecular elements for functionally blocking protein pores.
• This strategy effectively inhibits the AAA-ATPase p97, confirming substrate threading as its mechanism.
• The developed pore binders offer a new avenue for drug discovery and treating diseases linked to p97 dysfunction.