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abstracts
Precise tuning of gene expression by transcriptional regulators determines the response to internal and external chemical signals and adjusts the metabolic machinery for many cellular processes. As a part of ongoing efforts by the Midwest Center for Structural Genomics, a number of transcription factors were selected to study protein-ligand and protein-DNA interactions. HcaR, a new member of the MarR/SlyA family of transcription regulators from soil bacteria Acinetobacter sp. ADP1, is an evolutionarily atypical regulator and represses hydroxycinnamate (hca) catabolic genes. Hydroxycinnamates containing an aromatic ring play diverse, critical roles in plant architecture and defense. HcaR regulates the expression of the hca catabolic operon, allowing this and related bacterial strains to utilize hydroxycinnamates: ferulate, p-coumarate, and caffeate as sole sources of carbon and energy. HcaR appears to be capable of responding to multiple aromatic ligands. These aromatic compounds bind to HcaR and reduce its affinity to the specific DNA sites. As a result, the transcription of genes encoding several catabolic enzymes is up-regulated. The HcaR structures of the apo-form and in a complex with several ligands: ferulic acid, 3,4 dihydroxybenzoic acid, vanillin and p-coumaric acid have been determined to understand how HcaR accommodates various aromatic compounds using the same binding pocket. We also have identified a potential DNA site for HcaR in the regulatory region upstream of the genes of the hca catabolic operon in Acinetobacter sp. ADP1 and have confirmed DNA binding by EMSA. The co-crystal structure of HcaR and palindromic 24-mer DNA has been determined for this DNA site. The crystal structures of HcaR, the apo-form, ligand-bound forms, and the specific DNA-bound form provide critical structural basis of protein-ligand (substrates or product) and protein-DNA interactions to understand the regulation of the expression of hydroxycinnamate (hca) catabolic genes. Our studies allow for better understanding of DNA-binding and regulation by this important group of transcription factors belonging to the MarR/SlyA families. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
abstracts
The NrtR family of bacterial transcription factors is characterized by an N-terminal Nudix hydrolase-like effector binding domain and a C-terminal DNA binding domain. A bioinformatics analysis of the NrtR family represented by uncharacterized protein BT0354 in Bacteroides thetaiotaomicron suggests that these regulators control the catabolic pathways for L-arabinose. Many bacteria use L-arabinose as the sole source of carbon energy. The L-arabinose utilization pathway and its transcriptional regulation have been studied for a long time in several model microorganisms. Here we provide biochemical and structural characterization of the novel arabinose-responsive regulator of NrtR family protein BT0354, L-arabinose regulator from B. thetaiotaomicron (BtAraR). The BtAraR DNA binding and the role of effector molecule L-arabinose were confirmed using electrophoretic mobility shift assays. We have solved the crystal structures of BtAraR for two apo forms, and complexes with L-arabinose and double-stranded DNA target. The apo-1 form was solved as two dimers/AU in the R3 space group at 2.35 Å, while the apo-2 form was solved as one monomer/AU in the I213 space group at 2.56 Å resolution. The L-arabinose and DNA complex structures were solved as a dimer/AU in the P21 space group at 1.95 Å resolution and the P23 space group at 3.05 Å resolution, respectively. The biological unit of this protein is a dimer while the N-terminal ligand binding domain of the monomer adopts a Nudix hydrolase-like fold and the C-terminal DNA binding domain is a winged helix-turn-helix. The DNA binding-releasing mechanism can be rationalized through the comparison and analyses of these structures. The apo and DNA bound structures are more similar compared to the L-arabinose-bound structure. The r.m.s. deviation for the apo and DNA bound structures is 1.13 Å, while that for apo and the L-arabinose-bound structures is 4.54 Å. Details about the DNA binding mode, L-arabinose binding and L-arabinose induced structural change will be presented. This work was supported by National Institutes of Health grant GM094585 and by the U. S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
abstracts
The New Delhi Metallo β-lactamase (NDM-1), first identified in Klebsiella pneumoniae has been shown to hydrolyze nearly all clinical β-lactam antibiotics including carbapenems, considered "last resort" antibiotics. Its gene resides on mobile plasmids that move between different strains of bacteria posing a serious global threat to human health. There have also been reports of several variants, up to NDM-9, some with increased carbapenemase activity. As part of the NIGMS PSI:Biology effort, the Midwest Center for Structural Genomics (MCSG) together with the Structures of Mtb Proteins Conferring Susceptibility to Known Mtb Inhibitors partnership, made significant progress in investigating the enzyme atomic structure and catalytic mechanism. A large number of protein constructs as well as mutants were made and a number of high-resolution structures of NDM-1 (no Zn, one Zn, two Zn, two Mn or Cd, and complexed with antibiotics) and NDM-1 variants, NDM-2, NDM-3, NDM-4, NDM-5 and NDM-6 have been determined. We have determined the two structures of Michaelis complex: NDM-1 with two cadmium ions and a mixture of hydrolyzed and unhydrolyzed ampicillin (1.50 Å) and one with two cadmium ions and partly hydrolyzed faropenem (2.00 Å). The crystal structures revealed a ligand-binding pocket consisting of several flexible loops capable of accommodating many β-lactam substrates of different sizes and shapes. The structures with various metals suggest that the distance between the two metal atoms is closely correlated with substrate binding efficiency and hydrolysis and the pH-dependency of catalytic activity. For better understanding of catalytic mechanism of NDM-1, particularly the dynamics of substrate binding and the energy surfaces along the suggested reaction pathways, molecular dynamics calculations and hybrid classical/quantum (QM/MM) calculations were performed. This work was supported by NIH Grant GM094585 and by the U.S. DOE, OBER contract DE-AC02-06CH11357
