Biomedical subjects

John J Tanner

Publications and source records attributed to John J Tanner.

23 records · Page 2Linked to original sources

PubMed2004

Identification and characterization of the DNA-binding domain of the multifunctional PutA flavoenzyme.

The PutA flavoprotein from Escherichia coli is a transcriptional repressor and a bifunctional enzyme that regulates and catalyzes proline oxidation. PutA represses transcription of genes putA and putP by binding to the control DNA region of the put regulon. The objective of this study is to define and characterize the DNA binding domain of PutA. The DNA binding activity of PutA, a 1320 amino acid polypeptide, has been localized to N-terminal residues 1-261. After exploring a potential DNA-binding region and an N-terminal deletion mutant of PutA, residues 1-90 (PutA90) were determined to contain DNA binding activity and stabilize the dimeric structure of PutA. Cell-based transcriptional assays demonstrate that PutA90 functions as a transcriptional repressor in vivo. The dissociation constant of PutA90 with the put control DNA was estimated to be 110 nm, which is slightly higher than that of the PutA-DNA complex (K(d) approximately 45 nm). Primary and secondary structure analysis of PutA90 suggested the presence of a ribbon-helix-helix DNA binding motif in residues 1-47. To test this prediction, we purified and characterized PutA47. PutA47 is shown to purify as an apparent dimer, to exhibit in vivo transcriptional activity, and to bind specifically to the put control DNA. In gel-mobility shift assays, PutA47 was observed to bind cooperatively to the put control DNA with an overall dissociation constant of 15 nm for the PutA47-DNA complex. Thus, N-terminal residues 1-47 are critical for DNA-binding and the dimeric structure of PutA. These results are consistent with the ribbon-helix-helix family of transcription factors.

Amino Acid Sequence↗

PubMed2004

Detection of L-lactate in polyethylene glycol solutions confirms the identity of the active-site ligand in a proline dehydrogenase structure.

Polyethylene glycol (PEG) is often used in protein crystallography as a low-ionic-strength precipitant for crystallization and as a cryoprotectant for low-temperature data collection. Prompted by the discovery of an apparent L-lactate molecule bound in the active site of the Escherichia coli PutA proline dehydrogenase domain crystal structure, the L-lactate concentration of several PEG solutions was measured. 50%(w/v) solutions of PEGs with molecular weight 3000, 4000 and 8000 contain millimolar levels of L-lactate. In contrast, L-lactate was not detected in solutions of PEG monomethyl ethers or PEG 3350. These results help to explain why L-lactate was present in the proline dehydrogenase domain crystal structure. This work also has implications for the crystallization of enzymes that bind L-lactate.

Bacterial Proteins↗

PubMed2003

MRSAD: using anomalous dispersion from S atoms collected at Cu Kalpha wavelength in molecular-replacement structure determination.

The use of single-wavelength anomalous dispersion (SAD) from S atoms collected in-house to overcome model bias in molecular-replacement (MR) structure determination is demonstrated. The test case considered is a P6(5)22 anti-ssDNA Fab crystal with a theoretical anomalous signal of 0.8% and a diffraction limit of 2.3 A, from which a 360 degrees, 39-fold redundant data set was collected. A nearly complete anomalous scatterer substructure could be quickly built from anomalous difference Fourier analysis based on phases from a full or partial MR solution. The resulting SAD phases were improved with density modification and used to calculate an unbiased electron-density map that could be used for model building. This map displayed clear and continuous density for almost the entire main chain, as well as good density for most side chains. The favorable results obtained from this realistic test case suggest that anomalous differences from S atoms should be routinely collected and used in MR structure determination.

Copper↗

PubMed2003

Structure of the proline dehydrogenase domain of the multifunctional PutA flavoprotein.

The PutA flavoprotein from Escherichia coli plays multiple roles in proline catabolism by functioning as a membrane-associated bi-functional enzyme and a transcriptional repressor of proline utilization genes. The human homolog of the PutA proline dehydrogenase (PRODH) domain is critical in p53-mediated apoptosis and schizophrenia. Here we report the crystal structure of a 669-residue truncated form of PutA that shows both PRODH and DNA-binding activities, representing the first structure of a PutA protein and a PRODH enzyme from any organism. The structure is a domain-swapped dimer with each subunit comprising three domains: a helical dimerization arm, a 120-residue domain containing a three-helix bundle similar to that in the helix-turn-helix superfamily of DNA-binding proteins and a beta/alpha-barrel PRODH domain with a bound lactate inhibitor. Analysis of the structure provides insight into the mechanism of proline oxidation to pyrroline-5-carboxylate, and functional studies of a mutant protein suggest that the DNA-binding domain is located within the N-terminal 261 residues of E. coli PutA.

Bacterial Proteins↗

PubMed2002

A structurally conserved water molecule in Rossmann dinucleotide-binding domains.

A computational comparison of 102 high-resolution (</=1.90 A) enzyme-dinucleotide (NAD, NADP, FAD) complexes was performed to investigate the role of solvent in dinucleotide recognition by Rossmann fold domains. The typical binding site contains about 9-12 water molecules, and about 30% of the hydrogen bonds between the protein and the dinucleotide are water mediated. Detailed inspection of the structures reveals a structurally conserved water molecule bridging dinucleotides with the well-known glycine-rich phosphate-binding loop. This water molecule displays a conserved hydrogen-bonding pattern. It forms hydrogen bonds to the dinucleotide pyrophosphate, two of the three conserved glycine residues of the phosphate-binding loop, and a residue at the C-terminus of strand four of the Rossmann fold. The conserved water molecule is also present in high-resolution structures of apo enzymes. However, the water molecule is not present in structures displaying significant deviations from the classic Rossmann fold motif, such as having nonstandard topology, containing a very short phosphate-binding loop, or having alpha-helix "A" oriented perpendicular to the beta-sheet. Thus, the conserved water molecule appears to be an inherent structural feature of the classic Rossmann dinucleotide-binding domain.

Animals↗