AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on ...developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The extreme 5'-end of the enterovirus RNA genome contains a conserved cloverleaf-like domain that recruits 3CD and PCBP proteins required for initiating genome replication. Here, we report the ...crystal structure at 1.9 Å resolution of this domain from the CVB3 genome in complex with an antibody chaperone. The RNA folds into an antiparallel H-type four-way junction comprising four subdomains with co-axially stacked sA-sD and sB-sC helices. Long-range interactions between a conserved A40 in the sC-loop and Py-Py helix within the sD subdomain organize near-parallel orientations of the sA-sB and sC-sD helices. Our NMR studies confirm that these long-range interactions occur in solution and without the chaperone. The phylogenetic analyses indicate that our crystal structure represents a conserved architecture of enteroviral cloverleaf-like domains, including the A40 and Py-Py interactions. The protein binding studies further suggest that the H-shape architecture provides a ready-made platform to recruit 3CD and PCBP2 for viral replication.
RNA is highly negatively charged and often acquires complex structures that require the presence of divalent cations. Subtle changes in conformation resulting from changes in sequence can affect the ...way ions associate with RNA. Riboswitches are RNA molecules that are involved in the control of gene expression in bacteria and are excellent systems for testing the effects of sequence variations on the conformation of RNA because they contain a highly conserved binding pocket but present sequence variability among different organisms. In this work, we have compared the aptamer domain of a proposed M-box riboswitch from Mycobacterium tuberculosis with the aptamer domain of a validated M-box riboswitch from Bacillus subtilis. We have in vitro transcribed and purified wild-type (WT) M-box riboswitches from M. tuberculosis and B. subtilis as well as a variety of mutated aptamers in which regions from one riboswitch have been replaced with regions from the other riboswitch. We have used ultraviolet unfolding experiments and circular dichroism to characterize the interactions of WT and related M-box riboswitches with divalent cations. Our results show that M-box from M. tuberculosis associates with Mg2+ and Sr2+ in a similar fashion while M-box from B. subtilis discriminates between these two ions and appears to associate better with Mg2+. Our overall results show that M-box from M. tuberculosis interacts differently with cations than M-box from B. subtilis and suggest conformational differences between these two riboswitches.
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•CBPs and their targets deliver cell function(s) via oscillations in free Ca2+.•The BFF physiological framework for studying binding and allostery will be illustrated here with ...CBPs.•A type 1 BFF event is concerted within a physiologically relevant timeframe and a type 2 BFF mechanism is stepwise.
EF-hand Ca2+-binding proteins (CBPs), such as S100 proteins (S100s) and calmodulin (CaM), are signaling proteins that undergo conformational changes upon increasing intracellular Ca2+. Upon binding Ca2+, S100 proteins and CaM interact with protein targets and induce important biological responses. The Ca2+-binding affinity of CaM and most S100s in the absence of target is weak (CaKD > 1 μM). However, upon effector protein binding, the Ca2+ affinity of these proteins increases via heterotropic allostery (CaKD < 1 μM). Because of the high number and micromolar concentrations of EF-hand CBPs in a cell, at any given time, allostery is required physiologically, allowing for (i) proper Ca2+ homeostasis and (ii) strict maintenance of Ca2+-signaling within a narrow dynamic range of free Ca2+ ion concentrations, Ca2+free. In this review, mechanisms of allostery are coalesced into an empirical “binding and functional folding (BFF)” physiological framework. At the molecular level, folding (F), binding and folding (BF), and BFF events include all atoms in the biomolecular complex under study. The BFF framework is introduced with two straightforward BFF types for proteins (type 1, concerted; type 2, stepwise) and considers how homologous and nonhomologous amino acid residues of CBPs and their effector protein(s) evolved to provide allosteric tightening of Ca2+ and simultaneously determine how specific and relatively promiscuous CBP-target complexes form as both are needed for proper cellular function.
Wolbachiae are obligate intracellular bacteria that infect arthropods and certain nematodes. Usually maternally inherited, they may provision nutrients to (mutualism) or alter sexual biology of ...(reproductive parasitism) their invertebrate hosts. We report the assembly of closed genomes for two novel wolbachiae,
CfeT and
CfeJ, found co-infecting cat fleas (
) of the Elward Laboratory colony (Soquel, CA, USA).
CfeT is basal to nearly all described
supergroups, while
CfeJ is related to supergroups C, D and F. Both genomes contain laterally transferred genes that inform on the evolution of
host associations.
CfeT carries the Biotin synthesis Operon of Obligate intracellular Microbes (BOOM); our analyses reveal five independent acquisitions of BOOM across the
tree, indicating parallel evolution towards mutualism. Alternately,
CfeJ harbors a toxin-antidote operon analogous to the
Pip
operon recently characterized as an inducer of cytoplasmic incompatibility (CI) in flies.
CfeJ
and three adjacent genes are collectively similar to large modular toxins encoded in CI-like operons of certain
strains and
species, signifying that CI toxins streamline by fission of large modular toxins. Remarkably, the
.
genome itself contains two CI-like antidote genes, divergent from
CfeJ
, revealing episodic reproductive parasitism in cat fleas and evidencing mobility of CI loci independent of WO-phage. Additional screening revealed predominant co-infection (
CfeT/
CfeJ) amongst
.
colonies, though fleas in wild populations mostly harbor
CfeT alone. Collectively, genomes of
CfeT,
CfeJ, and their cat flea host supply instances of lateral gene transfers that could drive transitions between parasitism and mutualism.
Pathogenic Bacillus and clostridial (i.e., Clostridium and Clostridioides) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, ...which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-type complex composed of a catalytic subunit that is toxic (A) and an oligomeric cell-binding and delivery subunit (B), where upon delivery of A into the cytoplasm of the host cell it catalytically ADP-ribosylates actin and rapidly induces host cell death. In this review, binary toxins expressed by Bacillus thuringiensis, Clostridioides difficile, and Clostridium perfringens will be discussed, with particular focus placed upon the structural elucidations of their respective B subunits and how these findings help to deconvolute how toxic enzyme delivery into target host cells is achieved by these deadly bacteria.
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