Molecular Tweezers Block the Functional Pore of a Protein Machine

We present symmetric multivalent tweezers as the first class of supramolecular elements designed to cover and functionally block a protein pore. As a model, we chose the enzyme p97, a hexameric AAA-ATPase that unfolds or segregates substrate proteins by threading them through a pore and channel at the center of the symmetric p97 hexamer fueled by ATP hydrolysis. In a rational design approach, we developed a new class of p97 inhibitors, guided by molecular modeling. These dock onto lysine residues at the entry of the pore via appropriately positioned molecular tweezers. Ligand binding was accompanied by induction of fluorescence of the built-in binding sensitive luminophores which served as a sensor for affinity determination. We further confirmed specific interaction with p97 as well as concomitant inhibition of ATPase activity and protein substrate unfolding using an array of biophysical methods and state-of-the art biochemical assays. Specific binding was also validated by mutagenesis, demonstrating that inhibition of p97 function was mediated by blocking the pore entrance. Especially C₃-symmetric multivalent tweezers potently inhibited ATPase activity and protein substrate processing consistent with the symmetry of the docking site. Our data independently confirm substrate threading as a mechanism for protein unfolding by p97 and highlight multivalent tweezers as a supramolecular strategy to target pores in various proteins. Since p97 and related protein machines are vital for protein quality control and cell survival, the new pore binders may open a new approach to combat diseases and be employed in drug discovery.