Mechanism of active substrate delivery by the AAA+ protease adaptor CIpS

Abstract

AAA+ molecular machines power myriad cellular processes including protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication. Protein quality control in all organisms involves deployment of ATP-dependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel. Adaptor proteins assist in recognition and degradation of certain substrates, but how enzyme-adaptor pairs ensure proper substrate selection is incompletely understood. In this thesis I focus on the delivery mechanism employed by the bacterial adaptor protein CIpS. The CIpS adaptor collaborates with the AAA+ CIpAP protease to recognize and degrade N-end rule substrates. CIpS binds the substrate's N-degron and assembles into a high-affinity CIpS-substrate-CIpA complex, but how the N-degron is transferred from CIpS to the axial pore of the AAA+ CIpA unfoldase to initiate degradation is not known. Here, we demonstrate that the unstructured N-terminal extension (NTE) of CIpS enters the CIpA processing pore in the active ternary complex and that CIpA engagement of the CIpS NTE is crucial for CIpS-mediated substrate delivery. In addition, I report evidence that CIpA engagement of the CIpS NTE drives structural rearrangements in CIpS important for N-end rule substrate delivery. Furthermore, our preliminary experiments suggest that CIpS is able to resist degradation by CIpAP due to a combination of a high local stability and a challenging translocation sequence at the junction of the NTE and folded core domain. I propose a model in which CIpA remodels CIpS by translocating the NTE, triggering delivery of the N-end rule substrate. Similar mechanisms may be employed by other AAA+ enzymes that collaborate with adaptor proteins to remodel/disassemble substrates without destroying them by degradation.