The Brennan Lab's Research Focus
The overarching research focus of the Brennan Laboratory is to understand the molecular mechanisms of the fundamental processes of transcription and post transcription regulation. Our experimental approach to study these processes utilizes macromolecular crystallography and a wide variety of biochemical and biophysical methodologies. We have been fortunate to have determined a number of “first” structures of a multiple biologically important proteins and protein-nucleic acid complexes including those of the first LacI family member, PurR, bound to a cognate operator site, the first MerR family member, BmrR, bound to bmr promoter DNA, the first MarR family member, OhrR, bound to cognate DNA and the first full-length protein-phosphoprotein-DNA complex, CcpA-HPr(Ser46P) bound to a cre site. Each of these structures provided an atomic view of their DNA recognition mechanisms and helped unravel their roles in transcription regulation of biological processes such as de novo purine biosynthesis and carbon catabolite regulation as well as their induction mechanisms. We are still addressing key unresolved issues surrounding OhrR as well as other proteins and their germane complexes that are involved in sensing and detoxifying reactive oxygen species (ROS). Indeed, we are now engaged in a collaboration with John Helmann at Cornell University in order to understand the mechanism of a key enzyme found in bacilli and firmicutes that is involved in the biosynthesis of bacillithiol, a small molecule antioxidant, which appears to be functionally equivalent to glutathione. Further, our interest in sensing and reducing ROS in Neisseria has led to biochemical and structural studies on three crucial proteins, NmlR, MtrR and ng1427, which are transcriptional regulators of defence systems against the toxic effects of ROS.
Over the past decade we have also had a strong interest in understanding the mechanisms of multidrug resistance (mdr). The Brennan laboratory was the first to describe the structures of any multidrug-binding transcription repressor or activator, QacR and BmrR, respectively, in complex with multiple drugs. The QacR-drug structures revealed the multisite or minipocket mechanism by which a single protein, multidrug efflux pumps included, can bind multiple chemically and structurally dissimilar ligands and toxins. Indeed, we defined these pockets as polyspecific rather than nonspecific. The BmrR-drug complexes defined a second mechanism of multidrug binding, whereby the drug adjusts to a more “rigid” or fixed binding pocket. We continue our studies on several novel multidrug-binding proteins, including MepR from S. aureus, MtrR from N. gonorrhoeae, and NfxB from P. aeruginosa, with the goal of defining their molecular mechanisms of multidrug binding, induction and gene regulation. Our MepR and MtrR work are collaborations with Glenn Kaatz at Wayne State University School of Medicine, and Bill Shafer at Emory, respectively. More recently my group, in a long-standing collaboration with the laboratories of Maria Schumacher at Duke University and Kim Lewis at Northeastern University, has turned towards studying bacterial persistence, which results in multidrug tolerance and is the likely origin of recurrent, essentially incurable infections. Together we described the structures of the first bona fide E. coli persistence factor, HipA, and demonstrated it is a serine-threonine protein kinase and remarkably its novel mechanism of autophosphorylation (P-loop motif ejection) that results in inhibition of the kinase, which is a critical step to restart growth and hence infection. In order to understand the primary means of inhibition of this toxin by its antitoxin mate, HipB, we determined the structure of a HipA-HipB-DNA complex, which revealed HipB locks HipA into an open conformation and sequesters the toxin from any targets. HipB is also responsible for the autorepression of the hipBA operon. Currently, we are in the process of identifying HipA substrates, and hence any HipA consensus phosphorylation sequence, and determining the binding mechanisms and biochemical preferences of HipB for the four hipBA operator sites in the absence and in the presence of HipA. We have just begun pursuing the molecular and structural bases of persistence in other pathogenic bacteria, including Klebsiella, Shigella, Burkholderia, and Mycobacteria.
The Brennan laboratory has pursued its interest in post transcriptional regulation by carrying out structure-function studies on Hfq, an RNA chaperone that binds both small noncoding RNAs and their target mRNAs to effect gene expression. We have chosen Hfq proteins from both Gram-positive and Gram-negative bacteria and were the first to show the mechanisms that these proteins use to bind U-rich RNA as well as A-tract RNA. We continue our structural and biochemical studies on E coli, S. aureus and L. monocytogenes Hfq proteins bound to other large RNAs and DNA in order to delineate all sites of nucleic acid binding and the full function of Hfq.