Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo–electron microscopy to determine structures of the Yersinia pestis Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, “open” spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed “closed” conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.

In cells, dedicated AAA+ ATPases deposit hexameric, ring-shaped helicases onto DNA to initiate chromosomal replication. To better understand the mechanisms by which helicase loading can occur, we used cryo-EM to determine sub-4-Å -resolution structures of the E. coli DnaB,DnaC helicase,loader complex with nucleotide in pre-and post-DNA engagement states. In the absence of DNA, six DnaC protomers latch onto and crack open a DnaB hexamer using an extended N-terminal domain, stabilizing this conformation through nucleotide-dependent ATPase interactions. Upon binding DNA, DnaC hydrolyzes ATP, allowing DnaB to isomerize into a topologically closed, pre-translocation state competent to bind primase. Our data show how DnaC opens the DnaB ring and represses the helicase prior to DNA binding and how DnaC ATPase activity is reciprocally regulated by DnaB and DNA. Comparative analyses reveal how the helicase loading mechanism of DnaC parallels and diverges from homologous AAA+ systems involved in DNA replication and transposition.Supplemental Information includes seven figures and six videos and can be found with this article online at https://doi.

The AAA+ Lon protease is conserved from bacteria to humans, performs crucial roles in protein homeostasis, and is implicated in bacterial pathogenesis and human disease. We investigated how Lon selectively degrades specific substrates among a diverse array of potential targets. We report the discovery of HspQ as a new Lon substrate, unique specificity-enhancing factor, and potent allosteric activator. Lon recognizes HspQ via a C-terminal degron, whose precise presentation, in synergy with multipartite contacts with the native core of HspQ, is required for allosteric Lon activation. Productive HspQ-Lon engagement enhances degradation of multiple new and known Lon substrates. Our studies reveal the existence and simultaneous utilization of two distinct substrate recognition sites on Lon, an HspQ binding site and an HspQ-modulated allosteric site. Our investigations unveil an unprecedented regulatory use of an evolutionarily conserved heat shock protein and present a distinctive mechanism for how Lon protease achieves temporally enhanced substrate selectivity.

Through targeted inactivation of the ssrA and smpB genes, we establish that the trans-translation process is necessary for normal growth, adaptation to cellular stress, and virulence by the bacterial pathogen Francisella tularensis. The mutant bacteria grow slower, have reduced resistance to heat and cold shocks, and are more sensitive to oxidative stress and sub-lethal concentrations of antibiotics. Modifications of the tmRNA tag and use of higher resolution mass spectrometry approaches enabled the identification of a large number of native tmRNA substrates. Of particular significance to understanding the mechanism of trans-translation, we report the discovery of an extended tmRNA tag and extensive ladder-like pattern of endogenous protein tagging events in F. tularensis that are likely to be a universal feature of tmRNA activity in eubacteria. Furthermore, the structural integrity and the proteolytic function of the tmRNA tag are both crucial for normal growth and virulence of F. tularensis. Significantly, trans-translation mutants of F. tularensis are impaired in replication within macrophages and are avirulent in mouse models of tularemia. By exploiting these attenuated phenotypes, we find that the mutant strains provide effective immune protection in mice against lethal intradermal, peritoneal, and intranasal challenges with the fully virulent parental strain.