Evidence suggests that some nonsteroidal anti-inflammatory drugs (NSAIDs) possess antibacterial properties with an unknown mechanism. We describe the in vitro antibacterial properties of the NSAIDs carprofen, bromfenac, and vedaprofen, and show that these NSAIDs inhibit the Escherichia coli DNA polymerase III β subunit, an essential interaction hub that acts as a mobile tether on DNA for many essential partner proteins in DNA replication and repair. Crystal structures show that the three NSAIDs bind to the sliding clamp at a common binding site required for partner binding. Inhibition of interaction of the clamp loader and/or the replicative polymerase α subunit with the sliding clamp is demonstrated using an in vitro DNA replication assay. NSAIDs thus present promising lead scaffolds for novel antibacterial agents targeting the sliding clamp.

Protein-protein interactions based on linear motif (LM) recognition play roles in many cell regulatory processes. The E. coli sliding clamp is a protein mediator of replisome formation, which uses a common surface pocket composed of two subsites (I and II) to interact with LMs in multiple binding partners. A structural and thermodynamic dissection of sliding clamp-LM recognition has been performed, providing support for a sequential binding model. According to the model, a hydrophobic C-terminal LM dipeptide submotif acts as an anchor to establish initial contacts within subsite I, and this is followed by formation of a stabilizing hydrogen-bonding network between the flanking LM residues and subsite II. Differential solvation/desolvation during positioning of the submotifs is proposed as a driver for the sequential binding. Our model provides general insights into linear motif recognition and should guide the design of small-molecule inhibitors of the E. coli sliding clamp, an emerging antibacterial target.

The bacterial sliding clamp (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/ mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding clamp inhibitors as lead compounds with broad-spectrum antibacterial activity. Keywords CMMB

One Sentence Summary:Q forms a "nozzle" that narrows the RNA polymerase RNA-exit channel and extrudes ssRNA, preventing formation of RNA hairpins. Abstract:Lambdoid bacteriophage Q protein mediates the switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through transcription terminators preceding bacteriophage late genes. Q loads onto RNAP engaged in promoter-proximal pausing at a Q binding element (QBE) and an adjacent sigma-dependent pause element (SDPE) to yield a "Q-loading complex,"and Q subsequently translocates with RNAP as a pausing-deficient, termination-deficient "Q-loaded complex." Here, we report high-resolution structures of four states on the pathway of antitermination by Q from bacteriophage 21 (Q21): Q21, the Q21-QBE complex, the Q21-loading complex, and the Q21-loaded complex. The results show that Q21 forms a torus--a "nozzle"--that narrows and extends the RNAP RNA-exit channel, extruding single-stranded RNA and preventing formation of pause and terminator hairpins. Main Text:Lambdoid bacteriophage Q protein regulates gene expression through transcription antitermination, a mechanism of regulation that is widely used in bacteria and bacteriophage but that has been poorly understood (1-10; reviewed in 11-13).Q mediates the temporal switch from middle to late bacteriophage gene expression by enabling RNA polymerase (RNAP) to read through a transcription terminator preceding bacteriophage late genes ( Fig. 1A; 1-13). The Q-dependent gene regulatory cassette consists of the gene for Q followed by a transcription unit comprising a promoter (PR'), a promoter-proximal σ-dependent pause element (SDPE; a sequence resembling a promoter -10 element, at which σR2, the σ-factor module that recognizes promoter -10 elements, re-establishes sequence-specific protein-DNA interactions), and a terminator, followed by bacteriophage late genes. In the absence of Q, RNAP initiating transcription at the PR' promoter pauses at the SDPE and terminates at the terminator, and late genes are not expressed; in the presence of Q, RNAP initiating at the PR' promoter, escapes the SDPE and reads through the terminator, and late genes are expressed. 2Q is targeted to the PR' transcriptional unit through a multi-step process entailing: (i) formation of a "Q-loading complex" comprising a Q protein bound to a Q binding element (QBE) and a σ-containing transcription elongation complex (TEC) paused at the SDPE, and (ii) transformation into a "Q-loaded complex" comprising a Q-containing TEC that translocates processively, ignores pause elements, and ignores terminators (Fig. 1B; 3-13).Here, we report a set of four structures that define the structural basis of antitermination by Q from lambdoid bacteriophage Q21 (Q21; 11,14-15): i.e., (i) a crystal structure of Q21, (ii) a crystal structure of the Q21-QBE complex, (iii) a cryo-EM structure of the Q-loading complex, and (4) a cryo-EM structure of the Q-loaded complex.We solved a crystal structure of Q21 at 1.9 Å resolution by use of single...

The transcription factor, activator protein-1 (AP-1), binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic. A series of crystal structures of the AP-1 FosB/JunD bZIP domains reveal ordered DNA-binding regions in both FosB and JunD even in absence DNA. However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a ‘redox switch’ centered on an inter-molecular disulfide bond. Solution studies confirm that FosB/JunD cannot undergo structural transition and bind DNA when the redox-switch is in the ‘OFF’ state, and show that the mid-point redox potential of the redox switch affords it sensitivity to cellular redox homeostasis. The molecular and structural studies presented here thus reveal the mechanism underlying redox-regulation of AP-1 Fos/Jun transcription factors and provide structural insight for therapeutic interventions targeting AP-1 proteins.