Cryo-EM reveals the structure and dynamics of a 723-residue malate synthase G.

Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/ß domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy.

Condition-dependent structural collapse in the intrinsically disordered N-terminal domain of prion protein.

Prion protein is composed of a structure-unsolved N-terminal domain and a globular C-terminal domain. Under limited trypsin digestion, mouse recombinant prion protein can be cleaved into two parts at residue Lys105. Here, we termed these two fragments as the N-domain (sequence 23-105) and the C-domain (sequence 106-230). In this study, the structural properties of the N-domain, the C-domain, and the full-length protein were explored using small-angle X-ray scattering, analytical ultracentrifugation, circular dichroism spectroscopy, and the 8-anilino-1-naphthalenesulfonic acid binding assay. The conformation and size of the prion protein were found to change sensitively under the solvent conditions. The positive residues in the sequence 23-99 of the N-domain were found to be responsible for the enhanced flexibility with the salt concentration reduced below 5 mM. The C-domain containing a hydrophobic patch tends to unfold and aggregate during a salt-induced structural collapse. The N-domain collapsed together with the C-domain at pH 5.2, whereas it collapsed independently at pH 4.2. The positively charged cluster (sequence 100-105) in the N-domain contributed to protecting the exposed hydrophobic surface of the C-domain.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use.

BACKGROUND: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. METHODS: We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. RESULTS: We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric ß-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. CONCLUSIONS: Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy Aided by a Small Scaffold Protein.

Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in intrinsically noisy micrographs. One solution is to attach the target protein to a large protein scaffold to increase its apparent size and, therefore, image contrast. Here we report a novel scaffold design based on a trimeric helical protein, E. coli ornithine transcarbamylase (OTC), fused to human ubiquitin. As a proof of principle, we demonstrated the ability to resolve a cryo-EM map of a 26 kDa human ubiquitin C-terminal hydrolase (UCHL1) attached to the C-terminus of ubiquitin as part of the trimeric assembly. The results revealed conformational changes in UCHL1 upon binding to ubiquitin, namely, a significant displacement of α-helix 2, which was also observed by X-ray crystallography. Our findings demonstrated the potential of the trimeric OTC scaffold design for studying a large number of ubiquitin interacting proteins by cryo-EM.

The C-terminal D/E-rich domain of MBD3 is a putative Z-DNA mimic that competes for Zα DNA-binding activity.

The Z-DNA binding domain (Zα), derived from the human RNA editing enzyme ADAR1, can induce and stabilize the Z-DNA conformation. However, the biological function of Zα/Z-DNA remains elusive. Herein, we sought to identify proteins associated with Zα to gain insight into the functional network of Zα/Z-DNA. By pull-down, biophysical and biochemical analyses, we identified a novel Zα-interacting protein, MBD3, and revealed that Zα interacted with its C-terminal acidic region, an aspartate (D)/glutamate (E)-rich domain, with high affinity. The D/E-rich domain of MBD3 may act as a DNA mimic to compete with Z-DNA for binding to Zα. Dimerization of MBD3 via intermolecular interaction of the D/E-rich domain and its N-terminal DNA binding domain, a methyl-CpG-binding domain (MBD), attenuated the high affinity interaction of Zα and the D/E-rich domain. By monitoring the conformation transition of DNA, we found that Zα could compete with the MBD domain for binding to the Z-DNA forming sequence, but not vice versa. Furthermore, co-immunoprecipitation experiments confirmed the interaction of MBD3 and ADAR1 in vivo. Our findings suggest that the interplay of Zα and MBD3 may regulate the transition of the DNA conformation between B- and Z-DNA and thereby modulate chromatin accessibility, resulting in alterations in gene expression.

Covariate-adjusted heatmaps for visualizing biological data via correlation decomposition.

Motivation: Heatmap is a popular visualization technique in biology and related fields. In this study, we extend heatmaps within the framework of matrix visualization (MV) by incorporating a covariate adjustment process through the estimation of conditional correlations. MV can explore the embedded information structure of high-dimensional large-scale datasets effectively without dimension reduction. The benefit of the proposed covariate-adjusted heatmap is in the exploration of conditional association structures among the subjects or variables that cannot be done with conventional MV. Results: For adjustment of a discrete covariate, the conditional correlation is estimated by the within and between analysis. This procedure decomposes a correlation matrix into the within- and between-component matrices. The contribution of the covariate effects can then be assessed through the relative structure of the between-component to the original correlation matrix while the within-component acts as a residual. When a covariate is of continuous nature, the conditional correlation is equivalent to the partial correlation under the assumption of a joint normal distribution. A test is then employed to identify the variable pairs which possess the most significant differences at varying levels of correlation before and after a covariate adjustment. In addition, a z-score significance map is constructed to visualize these results. A simulation and three biological datasets are employed to illustrate the power and versatility of our proposed method. Availability and implementation: GAP is available to readers and is free to non-commercial applications. The installation instructions, the user's manual, and the detailed tutorials can be found at http://gap.stat.sinica.edu.tw/Software/GAP. Supplementary information: Supplementary Data are available at Bioinformatics online.

Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.

Galectins are ß-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their affinities for lactose, a common galectin inhibitor, vary significantly. Here, we aimed to examine the molecular basis of differential lactose affinities amongst galectins using solution-based techniques. Consistent dissociation constants of lactose binding were derived from nuclear magnetic resonance (NMR) spectroscopy, intrinsic tryptophan fluorescence, isothermal titration calorimetry and bio-layer interferometry for human galectin-1 (hGal1), galectin-7 (hGal7), and the N-terminal and C-terminal domains of galectin-8 (hGal8NTD and hGal8CTD, respectively). Furthermore, the dissociation rates of lactose binding were extracted from NMR lineshape analyses. Structural mapping of chemical shift perturbations revealed long-range perturbations upon lactose binding for hGal1 and hGal8NTD. We further demonstrated using the NMR-based hydrogen-deuterium exchange (HDX) that lactose binding increases the exchange rates of residues located on the opposite side of the ligand-binding pocket for hGal1 and hGal8NTD, indicative of allostery. Additionally, lactose binding induces significant stabilisation of hGal8CTD across the entire domain. Our results suggested that lactose binding reduced the internal dynamics of hGal8CTD on a very slow timescale (minutes and slower) at the expense of reduced binding affinity due to the unfavourable loss of conformational entropy.

A Multivalent Marine Lectin from Crenomytilus grayanus Possesses Anti-cancer Activity through Recognizing Globotriose Gb3.

In this study, we report the structure and function of a lectin from the sea mollusk Crenomytilus grayanus collected from the sublittoral zone of Peter the Great Bay of the Sea of Japan. The crystal structure of C. grayanus lectin (CGL) was solved to a resolution of 1.08 Å, revealing a ß-trefoil fold that dimerizes into a dumbbell-shaped quaternary structure. Analysis of the crystal CGL structures bound to galactose, galactosamine, and globotriose Gb3 indicated that each CGL can bind three ligands through a carbohydrate-binding motif involving an extensive histidine- and water-mediated hydrogen bond network. CGL binding to Gb3 is further enhanced by additional side-chain-mediated hydrogen bonds in each of the three ligand-binding sites. NMR titrations revealed that the three binding sites have distinct microscopic affinities toward galactose and galactosamine. Cell viability assays showed that CGL recognizes Gb3 on the surface of breast cancer cells, leading to cell death. Our findings suggest the use of this lectin in cancer diagnosis and treatment.

Solution structure and tandem DNA recognition of the C-terminal effector domain of PmrA from Klebsiella pneumoniae.

Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3(-)-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5'-CTTAAT-3' and 5'-CCTAAG-3', in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC-DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF-DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.

Uncovering the Mechanism of Forkhead-Associated Domain-Mediated TIFA Oligomerization That Plays a Central Role in Immune Responses.

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra ß-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

Crystal structure of vaccinia viral A27 protein reveals a novel structure critical for its function and complex formation with A26 protein.

Vaccinia virus envelope protein A27 has multiple functions and is conserved in the Orthopoxvirus genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21-84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner in vitro in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly in vitro. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.

Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal ß-hairpin conformation.

Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a ß-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, (1)H-(15)N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg(153) and the fragment K(170)KRK(173). We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

Co-modulated behavior and effects of differentially expressed miRNA in colorectal cancer.

BACKGROUND: MicroRNAs (miRNAs) are short noncoding RNAs (approximately 22 nucleotides in length) that play important roles in colorectal cancer (CRC) progression through silencing gene expression. Numerous dysregulated miRNAs simultaneously participate in the process of colon cancer development. However, the detailed mechanisms and biological functions of co-expressed miRNA in colorectal carcinogenesis have yet to be fully elucidated. RESULTS: The objective of this study was to identify the dysfunctional miRNAs and their target mRNAs using a wet-lab experimental and dry-lab bioinformatics approach. The differentially expressed miRNA candidates were identified from 2 miRNA profiles, and were confirmed in CRC clinical samples using reported target genes of dysfunctional miRNAs to perform functional pathway enrichment analysis. Potential target gene candidates were predicted by an in silico search, and their expression levels between normal and colorectal tumor tissues were further analyzed using real-time polymerase chain reaction (RT-PCR). CONCLUSION: Fifteen dysfunctional miRNAs were engaged in metastasis-associated pathways through comodulating 7 target genes, which were identified by using a multi-step approach. The roles of these candidate genes are worth further exploration in the progression of colon cancer, and could potentially be targets in future therapy.

dbDNV: a resource of duplicated gene nucleotide variants in human genome.

Gene duplications are scattered widely throughout the human genome. A single-base difference located in nearly identical duplicated segments may be misjudged as a single nucleotide polymorphism (SNP) from individuals. This imperfection is undistinguishable in current genotyping methods. As the next-generation sequencing technologies become more popular for sequence-based association studies, numerous ambiguous SNPs are rapidly accumulated. Thus, analyzing duplication variations in the reference genome to assist in preventing false positive SNPs is imperative. We have identified >10% of human genes associated with duplicated gene loci (DGL). Through meticulous sequence alignments of DGL, we systematically designated 1,236,956 variations as duplicated gene nucleotide variants (DNVs). The DNV database (dbDNV) (http://goods.ibms.sinica.edu.tw/DNVs/) has been established to promote more accurate variation annotation. Aside from the flat file download, users can explore the gene-related duplications and the associated DNVs by DGL and DNV searches, respectively. In addition, the dbDNV contains 304,110 DNV-coupled SNPs. From DNV-coupled SNP search, users observe which SNP records are also variants among duplicates. This is useful while ∼58% of exonic SNPs in DGL are DNV-coupled. Because of high accumulation of ambiguous SNPs, we suggest that annotating SNPs with DNVs possibilities should improve association studies of these variants with human diseases.

A unified framework of overlapping genes: towards the origination and endogenic regulation.

Overlapping genes are pairs of adjacent genes whose genomic regions partially overlap. They are notable by their potential intricate regulation, such as cis-regulation of nested gene-promoter configurations, and post-transcriptional regulation of natural antisense transcripts. The originations and consequent detailed regulation remain obscure. Herein, we propose a unified framework comprising biological classification rules followed by extensive analyses, namely, exon-sharing analysis, a human-mouse conservation study, and transcriptome analysis of hundreds of microarrays and transcriptome sequencing data (mRNA-Seq). We demonstrate that the tail-to-tail architecture would result from sharing functional elements in 3'-untranslated regions (3'-UTRs) of pre-existing genes. Dissimilarly, we illustrate that the other gene overlaps would originate from a new gene arising in a pre-existing gene locus. Interestingly, these types of coupled overlapping genes may influence each other synergistically or competitively during transcription, depending on the promoter configurations. This framework discloses distinctive characteristics of overlapping genes to be a foundation for a further comprehensive understanding of them.

MetaMirClust: discovery of miRNA cluster patterns using a data-mining approach.

Recent genome-wide surveys on ncRNA have revealed that a substantial fraction of miRNA genes is likely to form clusters. However, the evolutionary and biological function implications of clustered miRNAs are still elusive. After identifying clustered miRNA genes under different maximum inter-miRNA distances (MIDs), this study intended to reveal evolution conservation patterns among these clustered miRNA genes in metazoan species using a computation algorithm. As examples, a total of 15-35% of known and predicted miRNA genes in nine selected species constitute clusters under the MIDs ranging from 1kb to 50kb. Intriguingly, 33 out of 37 metazoan miRNA clusters in 56 metazoan genomes are co-conserved with their up/down-stream adjacent protein-coding genes. Meanwhile, a co-expression pattern of miR-1 and miR-133a in the mir-133-1 cluster has been experimentally demonstrated. Therefore, the MetaMirClust database provides a useful bioinformatic resource for biologists to facilitate the advanced interrogations on the composition of miRNA clusters and their evolution patterns.

Aberrant expression of miR-196a in gastric cancers and correlation with recurrence.

MicroRNAs (miRNAs) are short noncoding RNAs (~22 nt) that play important roles in the pathogenesis of human diseases by negatively regulating gene expression. Here, we examined the relationship between miR-196a and gastric cancer.By the analysis of 72 gastric cancer samples, we found that the expression level of miR-196a microRNA significantly increased in primary gastric cancer tissues versus adjacent normal tissues. In addition, extracellular miR-196a detected in conditioned medium was strongly correlated with its cellular expression status and increased circulating miR-196a in patient serum was associated with gastric cancer disease status and relapse. Furthermore, ectopic expression of miR-196a microRNA promoted the epithelial-mesenchymal transition and migration/invasion capabilities of transfected cells, suggesting its oncogenic potential in gastric cancer progression. Altogether, our data demonstrate that miR-196a exerts an oncogenic role in gastric cancer and miR-196a may be a novel biomarker for detecting gastric cancer and for monitoring disease recurrence.

Robust Design of Effective Allosteric Activators for Rsp5 E3 Ligase Using the Machine Learning Tool ProteinMPNN.

We used the deep learning tool ProteinMPNN to redesign ubiquitin (Ub) as a specific and functionally stimulating/enhancing binder of the Rsp5 E3 ligase. We generated 20 extensively mutated─up to 37 of 76 residues─recombinant Ub variants (UbVs), named R1 to R20, displaying well-folded structures and high thermal stabilities. These UbVs can also form stable complexes with Rsp5, as predicted using AlphaFold2. Three of the UbVs bound to Rsp5 with low micromolar affinity, with R4 and R12 effectively enhancing the Rsp5 activity six folds. AlphaFold2 predicts that R4 and R12 bind to Rsp5's exosite in an identical manner to the Rsp5-Ub template, thereby allosterically activating Rsp5-Ub thioester formation. Thus, we present a virtual solution for rapidly and cost-effectively designing UbVs as functional modulators of Ub-related enzymes.

Structural basis for the hydrolytic activity of the transpeptidase-like protein DpaA to detach Braun's lipoprotein from peptidoglycan.

IMPORTANCE: Cross-linking reaction of Braun's lipoprotein (Lpp) to peptidoglycan (PG) is catalyzed by some members of the YkuD family of transpeptidases. However, the exact opposite reaction of cleaving the Lpp-PG cross-link is performed by DpaA, which is also a YkuD-like protein. In this work, we determined the crystal structure of DpaA to provide the molecular rationale for the ability of the transpeptidase-like protein to cleave, rather than form, the Lpp-PG linkage. Our findings also revealed the structural features that distinguish the different functional types of the YkuD family enzymes from one another.

A 5+1 assemble-to-activate mechanism of the Lon proteolytic machine.

Many AAA+ (ATPases associated with diverse cellular activities) proteins function as protein or DNA remodelers by threading the substrate through the central pore of their hexameric assemblies. In this ATP-dependent translocating state, the substrate is gripped by the pore loops of the ATPase domains arranged in a universal right-handed spiral staircase organization. However, the process by which a AAA+ protein is activated to adopt this substrate-pore-loop arrangement remains unknown. We show here, using cryo-electron microscopy (cryo-EM), that the activation process of the Lon AAA+ protease may involve a pentameric assembly and a substrate-dependent incorporation of the sixth protomer to form the substrate-pore-loop contacts seen in the translocating state. Based on the structural results, we design truncated monomeric mutants that inhibit Lon activity by binding to the native pentamer and demonstrated that expressing these monomeric mutants in Escherichia coli cells containing functional Lon elicits specific phenotypes associated with lon deficiency, including the inhibition of persister cell formation. These findings uncover a substrate-dependent assembly process for the activation of a AAA+ protein and demonstrate a targeted approach to selectively inhibit its function within cells.