HcaR Ligand and DNA Interactions in the Regulation of Catabolic Gene Expression

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.

Structural studies of AraR from B. thetaiotaomicron

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.

Structure and specificity of novel aminopeptidase from marine sediment Archaea

The Earth's microbial diversity remained largely unexplored until recent developments in DNA sequencing. Novel methods enabled us to access genomic information of uncultured microbial organisms and create hypotheses about their metabolic capabilities. These predictions primarily rely on the sequence similarity between a novel protein and characterized proteins. Such an approach introduces a "culture" bias: the well-understood proteins come from a set of laboratory-grown bacteria, while novel microbial proteins are obtained from a variety of environments, including the most extreme. One such niche is ocean sediment – an unexplored ecosystem that plays important roles in geochemical cycles. Single-cell genomics targeting sedimentary populations identified four new archaeons encoding putative intra- and extra-cellular proteases [1]. This discovery suggests that heterotrophic marine Archaea evolved to degrade detrital proteins and might contribute to global carbon cycling. The novel proteases share some sequence similarity with well-known protein-degrading enzymes, but generally are distant homologs. Thus, functional screening is necessary to validate sequence-based predictions. One of the proteases shares sequence similarity with S15 peptidases, cocaine esterases and α-amino acid ester hydrolases (AEH). Phylogeny indicates that the gene is of bacterial origin. Enzymatic assays reveal α-aminopeptidase activity towards dipeptides with a preference for a small, L-configured hydrophobic residue at the N-terminus. The crystal structure shows a homotetrameric, self-compartmentalizing enzyme with four independent active sites localized inside the oligomeric assembly accessible from the internal channel. The active site contains a serine protease triad (Ser-His-Asp) and a cluster of negatively charged residues that bind the N-terminal NH3+ group of the substrate molecule. Therefore, the observed activity suggests that the enzyme (designated as AP TA1) may act on di- or tri-peptides produced during extracellular degradation and subsequently imported to the cell. As a close homolog of AEHs, it is also possible that AP TA1 might participate in the synthesis of yet-to-be-discovered secondary metabolites. Supported by NIH GM094585, DOE/BER DE-AC02-06CH11357 & C-DEBI 36202823 & 157595.

Carbapenemase NDM-1, Structural Analysis of the Catalytic Mechanism

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

Ultra-high resolution neutron and X-ray crystallography: structure of crambin

Neutron diffraction data to 1.1 Å was collected on a crystal of the small protein crambin at the Protein Crystallography Station (PCS) at Los Alamos, the highest resolution neutron structure of a protein to date, and a technical benchmark for the instrument. 95 % of the hydrogen atoms in the protein structure were resolved. The data allowed for the refinement of anisotropic temperature factors for selected deuterium atoms within the protein. Hydrogen bonding networks ambiguous in room temperature, ultra-high resolution (0.84 Å) electron density maps are clarified in the nuclear density maps. The ultra-high resolution data also reveals unusual H/D exchange patterns and novel chemistry in the side chains and protein backbone. Complementary X-ray diffraction data was collected at 19-ID at the Advanced Photon Source, with extensive re-configuration of the beamline to allow for operation at higher energy settings.