Structure of a small-molecule inhibitor of a DNA polymerase sliding clamp

Abstract

DNA polymerases attach to the DNA sliding clamp through a common overlapping binding site. We identify a small-molecule compound that binds the protein-binding site in the Escherichia coli ␤-clamp and differentially affects the activity of DNA polymerases II, III, and IV. To understand the molecular basis of this discrimination, the cocrystal structure of the chemical inhibitor is solved in complex with ␤ and is compared with the structures of Pol II, Pol III, and Pol IV peptides bound to ␤. The analysis reveals that the small molecule localizes in a region of the clamp to which the DNA polymerases attach in different ways. The results suggest that the small molecule may be useful in the future to probe polymerase function with ␤, and that the ␤-clamp may represent an antibiotic target.antibiotic target ͉ rational drug design ͉ fluorescence anisotropy ͉ crystallography T he replication machinery of all cells utilizes a ring-shaped, sliding-clamp protein that encircles DNA and slides along the duplex, thus acting as a mobile tether to hold the chromosomal replicase to DNA for high processivity (1-3). Sliding clamps from the three domains of life are remarkably similar in architecture (4-6). They consist of six domains of similar chain fold. The bacterial ␤-clamp is a homodimer, and each protomer consists of three domains, whereas eukaryotic and archaeal proliferating cell nuclear antigen (PCNA) clamps are homotrimers formed from protomers containing two domains each.Initially, ␤ and PCNA were identified as processivity factors for chromosomal replicases, but sliding clamps are now known to function with diverse DNA polymerases, repair factors, and cell cycle-control proteins (reviewed in ref.

Essential Biological Processes of an Emerging Pathogen: DNA Replication, Transcription, and Cell Division in Acinetobacter spp

“…An analogous mechanism is also shared by the PCNA sliding clamp of archaea and eukaryotes (209), although the consensus sequence recognized by PCNA (QxxLxxFF) is different from that of the bacterial ␤-binding motifs (55). The strong conservation of this protein-binding mechanism across bacteria, together with the essentiality of the interactions formed, makes the protein-binding groove of the ␤ sliding clamp ( the O'Donnell group has recently described a small molecule that binds within the ␤ clamp binding groove and is capable of disrupting key protein-protein interactions in diverse bacteria without inhibiting analogous interactions in the eukaryotic PCNA system (87). A second replication protein, the single-stranded DNAbinding protein (SSB), also interacts with many other proteins by way of a conserved peptide motif (229).…”

Structural Features and Functional Dependency on β-Clamp Define Distinct Subfamilies of Bacterial Mismatch Repair Endonuclease MutL

“…The assay was performed using a linear dsDNA as the substrate, as it is known that the termini of dsDNA enable the ␤-clamp to bind dsDNA without clamp loader (48). The 573-bp dsDNA was amplified, purified, and incubated with MutL proteins.…”

New Structural Templates for Clinically Validated and Novel Targets in Antimicrobial Drug Research and Development

“…In this context the bacterial replisome machinery, which consists of at least twelve interacting enzymes that are highly conserved in bacteria, is a good structure to target (Robinson et al 2012). Next to the DNA gyrase, which also belongs to the bacterial replisome and has already been discussed, the bacterial DnaN polymerase sliding clamp has recently attracted attention as an antibacterial target (Georgescu et al 2008b;Kjelstrup et al 2013;Wolff et al 2014;Yin et al 2014a;Yin et al 2014b;Holzgrabe 2015;Kling et al 2015;Yin et al 2015). The bacterial DnaN or β-clamp is a ring-shaped homodimer, with each monomer composed of three globular domains, that functions as a crucial subunit of the DNA polymerase III holoenzyme (Scheme 19).…”

“…O´Donnell an coworkers published the first non-peptidic small molecule inhibitor of the β-clamp, RU-7 58 (Scheme 21) (Georgescu et al 2008b). The compound was identified by screening a library of 30000 polar small molecules for their inhibito effe t o α-β2-binding by a fluorescence anisotropy titration assay.…”

“…It was demonstrated in further SAR studies in 2004 (Wijffels et al 2004) and 2011 (Wijffels et al 2011) that lipophilic N-terminal extensions of the pentapeptidic linear binding motif as well as substitutions with more lipophilic phenylalanine derivatives led to tremendous increases of the inhibitory effect of the i di g of the α-subunit of E. coli Pol III to the corresponding E. coli β-clamp (IC50 up to 20 nM). These linear motifs (LM) bind to the LM-binding pocket, which is highly conserved across Gram-negative and Gram-positive bacteria (Argiriadi et al 2006) and consists of two subsites (I and II) at the interface of the two domains on each mo o e of the β-clamp homodimer (Dalrymple et al 2001;Bunting et al 2003;Burnouf et al 2004;Georgescu et al 2008a;Georgescu et al 2008b). …”