Previous experiments have shown that this Phe is critical for SSB/ExoI complex formation (4, 6, 7)

Previous experiments have shown that this Phe is critical for SSB/ExoI complex formation (4, 6, 7). other SSB-interaction partners as well, which highlights their utility as reagents for investigating the roles of SSB/protein interactions in diverse DNA replication, recombination, and repair reactions. SSB), both of which play important roles in forming SSB/protein complexes (6). Interaction CNQX between the SSB-Ct and cellular genome maintenance machinery is essential in and, given the conservation of the SSB-Ct sequence among diverse bacterial SSBs, such interactions are likely to be common among bacteria (3). Tools that allow biochemical dissection of SSB/protein interactions would greatly facilitate experiments probing the diverse roles played by SSB in genome maintenance pathways. Open in a separate window Fig. 1. Small-molecule inhibitors disrupt ExoI/F-SSB-Ct complexes. (Exonuclease CNQX I (ExoI) bound to a peptide comprising the SSB-Ct sequence has provided a molecular model of SSB/protein interactions (4). In this structure, the C-terminal-most Phe of the SSB-Ct sequence forms a critical contact with ExoI in which the Phe side chain is enveloped in a hydrophobic pocket and its -carboxyl group is bound by an Arg side chain from ExoI (4). Intimate recognition of the SSB-Ct Phe appears to be a conserved feature in other SSB/protein interactions as well, and mutations that alter this residue in SSB are lethal to (4, 6C8). Roles for the acidic SSB-Ct residues in mediating interaction with ExoI have also been identified, leading to a model wherein SSB/ExoI association depends on multiple interactions for stability and specificity (6). The identification of this binding scheme has raised a number of questions as to the conservation of SSB-Ct binding sites among its many binding partners and the consequences of inhibiting interactions with SSB in reconstituted systems and in cells. To begin to answer these questions, we set out to develop a set of chemical tools to interrogate interactions between SSB and its protein partners. Here, we identify four small-molecule inhibitors that disrupt SSB/ExoI complexes. Two of these compounds have chemical structures that closely resemble the critical C-terminal Phe from the SSB-Ct element, indicating that they could act as peptide mimetics. Each of the inhibitors disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB stimulation of ExoI activity in nuclease reactions. Crystallographic and biochemical studies identify modes of inhibition for the compounds in which three of the molecules block SSB binding to ExoI by competitively binding to the SSB-Ct binding site on ExoI, whereas the fourth molecule appears to rely on allosteric effects to block SSB binding to ExoI. Remarkably, subsets of the compounds also dissociate complexes formed between the SSB-Ct element and two other SSB-interacting proteins (RecQ and PriA DNA helicases), indicating their utility as general SSB/protein complex inhibitors. Together, these studies provide unique biochemical tools for probing the roles of SSB/protein interactions. Results Identification of SSB/ExoI Interaction Inhibitors. A library of 50,400 small-molecule compounds was screened using a high-throughput fluorescence polarization (FP) assay to identify inhibitors that dissociate the complex created between SSB and ExoI, a well-studied SSB-binding partner. CNQX The assay monitored whether the addition of individual small molecules influences binding of a fluorescein-labeled SSB-Ct peptide (F-SSB-Ct) to ExoI by measuring the FP of F-SSB-Ct (FP is definitely 200?mP when bound CNQX to ExoI and 40?mP when free) (4). Our display identified more than 400 compounds that lowered FP ideals to 40?mP. The majority of these compounds were disregarded because of the intrinsic fluorescence or fluorescence-quenching properties or because of the common recognition as false positive hits in additional high-throughput FP screens. After screening the dose-dependent activity of the remaining compounds, four were pursued further (referred to as CFAM, BCBP, BOTP, and MPTA, Table?1). Interestingly, two of the compounds (BOTP and MPTA) experienced phenyl and carboxyl organizations structured around chiral carbons in related positions to analogous organizations from your C-terminal-most Phe of the SSB-Ct. Earlier experiments have shown that this Phe is critical for SSB/ExoI complex formation (4, 6, 7). These features could consequently be related to the abilities of the compounds to block ExoI/F-SSB-Ct complex formation. Table 1. Constructions, IC50, Kvalues with no measureable effect on ideals for the compounds assorted from 26??4?M for the most potent (CFAM) to 163??33?M for the least (MPTA) (Table?1). To determine whether the compounds could run by combined inhibition, in which the inhibitors can bind to either free ExoI or to the ExoI/substrate complex,.The assay monitored whether the addition of individual small molecules influences binding of a fluorescein-labeled SSB-Ct peptide (F-SSB-Ct) to ExoI by measuring the FP of F-SSB-Ct (FP is 200?mP when bound to ExoI and 40?mP when free) (4). of SSB/protein relationships in diverse DNA replication, recombination, and restoration reactions. SSB), both of which play important roles in forming SSB/protein complexes (6). Connection between the SSB-Ct and cellular genome maintenance machinery is essential in and, given the conservation of the SSB-Ct sequence among varied bacterial SSBs, such relationships are likely to be common among bacteria (3). Tools that allow biochemical dissection of SSB/protein interactions would greatly facilitate experiments probing the varied roles played by SSB in genome maintenance pathways. Open in a separate windows Fig. 1. Small-molecule inhibitors disrupt ExoI/F-SSB-Ct complexes. (Exonuclease I (ExoI) bound to a peptide comprising the SSB-Ct sequence has offered a molecular model of SSB/protein interactions (4). With this structure, the C-terminal-most Phe of the SSB-Ct sequence forms a critical contact with ExoI in which the Phe part chain CNQX is definitely enveloped inside a hydrophobic pocket and its -carboxyl group is definitely bound by an Arg part chain from ExoI (4). Romantic recognition of the SSB-Ct Phe appears to be a conserved feature in additional SSB/protein interactions as well, and mutations that alter this residue in SSB are lethal to (4, 6C8). Functions for the acidic SSB-Ct IKK-gamma (phospho-Ser85) antibody residues in mediating connection with ExoI have also been identified, leading to a model wherein SSB/ExoI association depends on multiple relationships for stability and specificity (6). The recognition of this binding scheme offers raised a number of questions as to the conservation of SSB-Ct binding sites among its many binding partners and the consequences of inhibiting relationships with SSB in reconstituted systems and in cells. To begin to solution these questions, we set out to develop a set of chemical tools to interrogate relationships between SSB and its protein partners. Here, we determine four small-molecule inhibitors that disrupt SSB/ExoI complexes. Two of these compounds have chemical structures that closely resemble the crucial C-terminal Phe from your SSB-Ct element, indicating that they could act as peptide mimetics. Each of the inhibitors disrupts ExoI/SSB-Ct peptide complexes and abrogates SSB activation of ExoI activity in nuclease reactions. Crystallographic and biochemical studies identify modes of inhibition for the compounds in which three of the molecules block SSB binding to ExoI by competitively binding to the SSB-Ct binding site on ExoI, whereas the fourth molecule appears to rely on allosteric effects to block SSB binding to ExoI. Amazingly, subsets of the compounds also dissociate complexes created between the SSB-Ct element and two additional SSB-interacting proteins (RecQ and PriA DNA helicases), indicating their power as general SSB/protein complex inhibitors. Collectively, these studies provide unique biochemical tools for probing the functions of SSB/protein interactions. Results Recognition of SSB/ExoI Connection Inhibitors. A library of 50,400 small-molecule compounds was screened using a high-throughput fluorescence polarization (FP) assay to identify inhibitors that dissociate the complex created between SSB and ExoI, a well-studied SSB-binding partner. The assay monitored whether the addition of individual small molecules influences binding of a fluorescein-labeled SSB-Ct peptide (F-SSB-Ct) to ExoI by measuring the FP of F-SSB-Ct (FP is definitely 200?mP when bound to ExoI and 40?mP when free) (4). Our display identified more than 400 compounds that lowered FP ideals to 40?mP. The majority of these compounds were disregarded because of the intrinsic fluorescence or fluorescence-quenching properties or because of the common recognition as false positive hits in additional high-throughput FP screens. After screening the dose-dependent activity of the remaining compounds, four were pursued further (referred to as CFAM, BCBP, BOTP, and MPTA, Table?1). Interestingly, two of the compounds (BOTP and MPTA) experienced phenyl and carboxyl organizations structured around chiral carbons in related positions to analogous organizations from your C-terminal-most Phe of the SSB-Ct. Earlier experiments have shown that this Phe is critical for SSB/ExoI complex formation (4, 6, 7). These features could consequently be related to the abilities of the compounds to block ExoI/F-SSB-Ct complex formation. Table 1. Constructions, IC50, Kvalues with no measureable.