Biomedical subjects

Steve Grkovic

Publications and source records attributed to Steve Grkovic.

5 recordsLinked to original sources

PubMed2006

New insights into BS69 functions.

The BS69 protein has been commonly described as a co-repressor associated with various transcription factors. However, this hypothesis relied predominantly on overexpression of tagged proteins due to the lack of a reliable BS69 antibody. We present for the first time a complete sequence of BS69 and valuable tools to characterize the endogenous protein. We show that the full-length BS69 protein, as well as minor alternatively spliced isoforms, is ubiquitously expressed, nuclear, and associates with chromatin and mitotic chromosomes. Accordingly, BS69 interacts with a set of chromatin remodeling factors, including ATP-dependent helicases, histone deacetylases, and histone methyltransferases, as well as the E2F6 transcription factor. These data strengthen a role for BS69 in gene repression and link BS69 to chromatin remodeling.

Alternative Splicing↗

PubMed2003

Interactions of the QacR multidrug-binding protein with structurally diverse ligands: implications for the evolution of the binding pocket.

The QacR multidrug-binding repressor protein regulates the expression of the Staphylococcus aureus qacA gene, a multidrug resistance (MDR) locus that is prevalent in clinical isolates of this important human pathogen. In this paper we demonstrate that the range of structurally diverse compounds capable of inducing qacA transcription is significantly more varied than previously appreciated, particularly in relation to bivalent cations. For all of the newly identified inducing compounds, induction of qacAexpression was correlated with a matching ability to dissociate QacR from operator DNA. Development of a ligand-binding assay based on intrinsic tryptophan fluorescence permitted dissociation constants to be determined for the majority of known QacR ligands, with values ranging from 0.2 to 82 microM. High-affinity binding of a compound to QacR in vitro was not found to correlate very strongly with either its in vivo inducing abilities or its structure. The latter observation indicated that the QacR ligand-binding pocket appears to have evolved to accommodate a wide range of toxic hydrophobic cations, rather than a specific class of compound. Importantly, the antimicrobial ligands of QacR included several plant alkaloids that share structural similarities with synthetic MDR substrates. This is consistent with the suggestion that the qacA-qacR MDR locus was recently derived from genes that protect against natural antimicrobial compounds.

ATP-Binding Cassette Transporters↗

PubMed2003

Stable low-copy-number Staphylococcus aureus shuttle vectors.

A series of Staphylococcus aureus-Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.

Bacterial Proteins↗

PubMed2002

Structural basis for cooperative DNA binding by two dimers of the multidrug-binding protein QacR.

The Staphylococcus aureus multidrug-binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by multiple structurally dissimilar drugs. QacR is a member of the TetR/CamR family of transcriptional regulators, which share highly homologous N-terminal DNA-binding domains connected to seemingly non-homologous ligand-binding domains. Unlike other TetR members, which bind approximately 15 bp operators, QacR recognizes an unusually long 28 bp operator, IR1, which it appears to bind cooperatively. To elucidate the DNA-binding mechanism of QacR, we determined the 2.90 A resolution crystal structure of a QacR-IR1 complex. Strikingly, our data reveal that the DNA recognition mode of QacR is distinct from TetR and involves the binding of a pair of QacR dimers. In this unique binding mode, recognition at each IR1 half-site is mediated by a complement of DNA contacts made by two helix-turn-helix motifs. The inferred cooperativity does not arise from cross-dimer protein-protein contacts, but from the global undertwisting and major groove widening elicited by the binding of two QacR dimers.

Bacterial Proteins↗

PubMed2002

Regulation of bacterial drug export systems.

The active transport of toxic compounds by membrane-bound efflux proteins is becoming an increasingly frequent mechanism by which cells exhibit resistance to therapeutic drugs. This review examines the regulation of bacterial drug efflux systems, which occurs primarily at the level of transcription. Investigations into these regulatory networks have yielded a substantial volume of information that has either not been forthcoming from or complements that obtained by analysis of the transport proteins themselves. Several local regulatory proteins, including the activator BmrR from Bacillus subtilis and the repressors QacR from Staphylococcus aureus and TetR and EmrR from Escherichia coli, have been shown to mediate increases in the expression of drug efflux genes by directly sensing the presence of the toxic substrates exported by their cognate pump. This ability to bind transporter substrates has permitted detailed structural information to be gathered on protein-antimicrobial agent-ligand interactions. In addition, bacterial multidrug efflux determinants are frequently controlled at a global level and may belong to stress response regulons such as E. coli mar, expression of which is controlled by the MarA and MarR proteins. However, many regulatory systems are ill-adapted for detecting the presence of toxic pump substrates and instead are likely to respond to alternative signals related to unidentified physiological roles of the transporter. Hence, in a number of important pathogens, regulatory mutations that result in drug transporter overexpression and concomitant elevated antimicrobial resistance are often observed.

Bacterial Proteins↗