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