Nanotools in biomedicine open up novel applications in research, diagnostics, and clinical care.

Kinetochores are chromatin‐bound multi‐protein complexes that allow high‐fidelity chromosome segregation during mitosis and meiosis. Kinetochore assembly is exclusively initiated at chromatin containing Cse4/CENP‐A nucleosomes. The molecular mechanisms ensuring that subcomplexes assemble efficiently into kinetochores only at centromeres, but not anywhere else, are incompletely understood. Here, we combine biochemical and genetic experiments to demonstrate that auto‐inhibition of the conserved kinetochore subunit Mif2/CENP‐C contributes to preventing unscheduled kinetochore assembly in budding yeast cells. We show that wild‐type Mif2 is attenuated in its ability to bind a key downstream component in the assembly pathway, the Mtw1 complex, and that addition of Cse4 nucleosomes overcomes this inhibition. By exchanging the N‐terminus of Mif2 with its functional counterpart from Ame1/CENP‐U, we have created a Mif2 mutant which bypasses the Cse4 requirement for Mtw1 binding in vitro , thereby shortcutting kinetochore assembly. Expression of this Mif2 mutant in cells leads to mis‐localization of the Mtw1 complex and causes pronounced chromosome segregation defects. We propose that auto‐inhibition of Mif2/CENP‐C constitutes a key concept underlying the molecular logic of kinetochore assembly.

The AAA+ ATPase p97/VCP together with different sets of substrate-delivery adapters and accessory cofactor proteins unfolds ubiquitinated substrates to facilitate degradation by the proteasome. The UBXD1 cofactor is connected to p97-associated multisystem proteinopathy but its biochemical function and structural organization on p97 has remained largely elusive. Using a combination of crosslinking mass spectrometry and biochemical assays, we identify an extended UBX (eUBX) module in UBXD1 related to a lariat in another cofactor, ASPL. Of note, the UBXD1-eUBX intramolecularly associates with the PUB domain in UBXD1 close to the substrate exit pore of p97. The UBXD1 PUB domain can also bind the proteasomal shuttling factor HR23b via its UBL domain. We further show that the eUBX domain has ubiquitin binding activity and that UBXD1 associates with an active p97-adapter complex during substrate unfolding. Our findings suggest that the UBXD1-eUBX module receives unfolded ubiquitinated substrates after they exit the p97 channel and before hand-over to the proteasome. The interplay of full-length UBXD1 and HR23b and their function in the context of an active p97:UBXD1 unfolding complex remains to be studied in future work.

AAA+ ATPase p97/valosin-containing protein (VCP)/Cdc48 is a key player in various cellular stress responses in which it unfolds ubiquitinated proteins to facilitate their degradation by the proteasome. P97 works in different cellular processes using alternative sets of cofactors and is implicated in multiple degenerative diseases. Ubiquitin regulatory X domain protein 1 (UBXD1) has been linked to pathogenesis and is unique amongst p97 cofactors because it interacts with both termini of p97. Its N-domain binds to the N-domain and N/D1 interface of p97 and regulates its ATPase activity. The PUB (peptide:N-glycanase and UBA or UBX-containing proteins) domain binds the p97 C-terminus, but how it controls p97 function is still unknown. Here we present the NMR structure of UBXD1-PUB together with binding studies, mutational analysis, and a model of UBXD1-PUB in complex with the p97 C-terminus. While the binding pocket is conserved among PUB domains, UBXD1-PUB features a unique loop and turn regions suggesting a role in coordinating interaction with downstream regulators and substrate processing

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.