Functional roles of the pre-sensor I insertion sequence in an AAA+ bacterial enhancer binding protein

Molecular machines belonging to the AAA+ superfamily of ATPases use NTP hydrolysis to remodel their versatile substrates. The presence of an insertion sequence defines the major phylogenetic presensor I insertion (pre-Sli) AAA+ superclade. In the bacterial σ⁵⁴-dependent enhancer binding protein phage shock protein F (PspF) the pre-Sli loop adopts different conformations depending on the nucleotidebound state. Single amino acid substitutions within the dynamic pre-Sli loop of PspF drastically change the ATP hydrolysis parameters, indicating a structural link to the distant hydrolysis site. We used a sitespecific protein-DNA proximity assay to measure the contribution of the pre-Sli loop in σ⁵⁴-dependent transcription and demonstrate that the pre-Sli loop is a major structural feature mediating nucleotide statedependent differential engagement with Eσ⁵⁴. We suggest that much, if not all, of the action of the pre-Sli loop is mediated through the L1 loop and relies on a conserved molecular switch, identified in a crystal structure of one pre-Sli variant and in accordance with the high covariance between some pre-Sli residues and distinct residues outside the pre-Sli sequence.