The flexible attachment of the N-domains to the ClpA ring body allows their use on demand.

ClpA is an Hsp100 chaperone that uses the chemical energy of ATP to remodel various protein substrates to prepare them for degradation. It comprises two AAA+ modules and the N-domain, which is attached N-terminally to the first AAA+ module through a linker. On the basis of cryo-electron microscopic and X-ray crystallographic data it has been suggested that the linker confers mobility to the N-domain. In order to define the role of the N-domain in ClpAP-dependent substrate degradation we have generated a Delta N variant at the protein level by introducing a protease cleavage site. The ClpA molecule generated in this way lacks the N-domain and the associated linker but is impaired only slightly in the processing of substrates that are degraded independently of ClpS. In fact, it shows increased catalytic efficiency in the degradation of ssrA-tagged GFP compared to ClpAwt. The role of the linker attaching the N-domain to the bulk of the molecule was probed by characterizing variants with different lengths of the linker. The degradation efficiency of a ClpS-dependent N-end rule substrate, FRliGFP, is reduced for linkers that are shorter or longer than natural linkers but remains the same for the variant where the linker is replaced by an engineered sequence of equivalent length. These results suggest that the flexible attachment of the N-domains to ClpA allows their recruitment to the pore on demand for certain substrates, while allowing them to move out of the way for substrates binding directly to the pore.