SAVED by a toxin: Structure and function of the CRISPR Lon protease

SummaryCRISPR antiviral defense systems such as the well-known DNA-targeting Cas9- and the more complex RNA-targeting type III systems are widespread in bacteria and archea 1, 2. The type III systems can orchestrate a complex antiviral response that is initiated by the synthesis of cyclic oligoadenylates (cOAs) upon foreign RNA recognition 3–5. These second messenger molecules bind to the CARF (CRISPR associated Rossmann-fold) domains of dedicated effector proteins that are often DNAses, RNAses, or putative transcription factors 6. The activated effectors interfere with cellular pathways of the host, inducing cell death or a dormant state of the cell that is better suited to avoid propagation of the viral attack 7, 8. Among a large set of proteins that were predicted to be linked to the type III systems 9, 10, the CRISPR-Lon protein caught our attention. The protein was predicted to be an integral membrane protein containing a SAVED-instead of a CARF-domain as well as a Lon protease effector domain. Here, we report the crystal structure of CRISPR-Lon. The protein is a soluble monomer and indeed contains a SAVED domain that accommodates cA4. Further, we show that CRISPR-Lon forms a stable complex with the 34 kDa CRISPR-T protein. Upon activation by cA4, CRISPR-Lon specifically cleaves CRISRP-T, releasing CRISPR-T23, a 23 kDa fragment that is structurally very similar to MazF toxins and is likely a sequence specific nuclease. Our results describe the first cOA activated proteolytic enzyme and provide the first example of a SAVED domain connected to a type III CRISPR defense system. The use of a protease as a means to unleash a fast response against a threat has intriguing parallels to eukaryotic innate immunity.

Cryo-EM structure of hexameric yeast Lon (PIM1) highlights importance of conserved structural elements

Lon protease is a conserved ATP-dependent serine protease composed of an AAA+ domain that mechanically unfolds substrates and a serine protease domain that degrades unfolded substrates. In yeast, dysregulation of Lon protease (PIM1) attenuates lifespan and leads to gross mitochondrial morphologic perturbations. Although structures of bacterial and human Lon protease reveal a hexameric assembly, PIM1 was speculated to form a heptameric assembly, and is uniquely characterized by a $\sim$50 residue insertion between the ATPase and protease domains. To understand the yeast-specific properties of PIM1, we determined a high-resolution cryo-EM structure of PIM1 in a substrate-translocating state. Here, we reveal that PIM1 forms a hexamer, conserved with that of bacterial and human Lon proteases, wherein the ATPase domains form a canonical closed spiral that enables pore loop residues to translocate substrate to the protease chamber. In the substrate-translocating state, PIM1 protease domains form a planar protease chamber in an active conformation and are uniquely characterized by a $\sim$15 residue C-terminal extension. These additional C-terminal residues form an alpha-helix that is located along the base of the protease domain. Finally, we did not observe density for the yeast-specific insertion between the ATPase and protease domains, likely due to high conformational flexibility. Biochemical studies to investigate the insertion using constructs that truncated or replaced the insertion with a glycine-serine linker suggest that the yeast-specific insertion is dispensable for PIM1 enzymatic function. Altogether, our structural and biochemical studies highlight unique components of PIM1 machinery and demonstrate evolutionary conservation of Lon protease function.

Essential roles of Lon protease in the morpho-physiological traits of the rice pathogen Burkholderia glumae

The highly conserved ATP-dependent Lon protease plays important roles in diverse biological processes. The lon gene is usually nonessential for viability; however, lon mutants of several bacterial species, although viable, exhibit cellular defects. Here, we show that a lack of Lon protease causes pleiotropic effects in the rice pathogen Burkholderia glumae. The null mutation of lon produced three colony types, big (BLONB), normal (BLONN), and small (BLONS), in Luria–Bertani (LB) medium. Colonies of the BLONB and BLONN types were re-segregated upon subculture, while those of the BLONS type were too small to manipulate. The BLONN type was chosen for further studies, as only this type was fully genetically complemented. BLONN-type cells did not reach the maximum growth capacity, and their population decreased drastically after the stationary phase in LB medium. BLONN-type cells were defective in the biosynthesis of quorum sensing (QS) signals and exhibited reduced oxalate biosynthetic activity, causing environmental alkaline toxicity and population collapse. Addition of excessive N-octanoyl-homoserine lactone (C8-HSL) to BLONN-type cell cultures did not fully restore oxalate biosynthesis, suggesting that the decrease in oxalate biosynthesis in BLONN-type cells was not due to insufficient C8-HSL. Co-expression of lon and tofR in Escherichia coli suggested that Lon negatively affects the TofR level in a C8-HSL-dependent manner. Lon protease interacted with the oxalate biosynthetic enzymes, ObcA and ObcB, indicating potential roles for the oxalate biosynthetic activity. These results suggest that Lon protease influences colony morphology, growth, QS system, and oxalate biosynthesis in B. glumae.

The Herbal Compound Thymol Targets Multiple Salmonella Typhimurium Virulence Factors for Lon Protease Degradation

Many important bacterial pathogens are using the type III secretion system to deliver effectors into host cells. Salmonella Typhimurium (S. Typhimurium) is a pathogenic Gram-negative bacterium with the type III secretion system as its major virulence factor. Our previous studies demonstrated that thymol, a monoterpene phenol derivative of cymene, inhibited S. Typhimurium invasion into mammalian cells and protected mice from infection. However, the antibacterial mechanism of thymol is not clear. In this study, we revealed that thymol interferes with the abundance of about 100 bacterial proteins through proteomic analysis. Among the 42 proteins whose abundance was reduced, 11 were important virulence factors associated with T3SS-1. Further analyses with SipA revealed that thymol directly interacts with this protein to induce conformational changes, which makes it susceptible to the Lon protease. In agreement with this observation, thymol effectively blocks cell invasion by S. Typhimurium. Thus, thymol represents a class of anti-virulence compounds that function by targeting pathogenic factors for degradation.