Recombinant Protein Expression, Crystallization, and Biophysical Studies of a <em>Bacillus</em>-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Yonsei University

The MarR family of transcriptional regulatory proteins in bacteria and archaea respond to environmental changes and regulate transcriptional processes by ligand binding or cysteine oxidation. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistances, causing a growing health problem. Therefore, it has been of great interest to locate the ligand binding site of MepR and reveal the ligand-mediated transcriptional regulation mechanism. Here, we report on the crystal structure of Bacillus cereus MepR-like transcription factor, BC0657, at 2.16 Å resolution. Interestingly, BC0657 was complexed with fortuitous pseudo-ligands, which were assessed to be lipid molecules containing a long fatty acid, rather than phenolic compounds previously observed in other MarR proteins. The BC0657-ligand interaction provides the first molecular view of how MepR recognizes ligands to respond to toxic chemicals. Moreover, our comparative structure analyses of ligand binding sites on BC0657 and its homologs suggest that transcriptional repression by MepR would be relieved by ligand-induced changes in dimerization organization.

Structure analysis of Bacillus cereus MepR-like transcription regulator, BC0657, in complex with pseudo-ligand molecules.

Transcription factors of the MarR family respond to internal and external changes and regulate a variety of biological functions through ligand association with microorganisms. MepR belongs to the MarR family, and its mutations are associated with the development of multidrug resistance in Staphylococcus aureus, which has caused a growing health problem. In this study, a Bacillus cereus MepR-like transcription regulator, BC0657, was crystallized. The BC0657 crystals diffracted to 2.05 Å resolution and belonged to either space group P6(2)22 or P6(4)22, with unit-cell parameters a = 110.57, b = 110.57, c = 67.29 Å. There was one molecule per asymmetric unit. Future comparative structural studies on BC0657 would extend knowledge of ligand-induced transcriptional regulatory mechanisms in the MarR family and would make a significant contribution to the design of antibiotic drugs against multidrug-resistant bacteria.

Purification, crystallization and X-ray crystallographic studies of a Bacillus cereus MepR-like transcription factor, BC0657.

BA1544 from Bacillus anthracis was previously annotated as a transcription factor for the gene cluster ba1554 - ba1558, but has not been experimentally characterized. B. anthracis is an obligate pathogen causing fatal inhalational anthrax, and BA1544 is absolutely conserved in Bacillus species, including Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides, with 100% sequence identity. To address the function of BA1544, we performed structural and biochemical studies, which revealed that BA1544 is a MazG protein. Thus, herein, the protein is defined as Bacillus-conserved MazG (BcMazG). Like other MazG structures, BcMazG assembles into a tetrameric architecture. Each monomer adopts a four-α-helix bundle that accommodates a metal ion using four acidic residues, and presents one putative substrate-binding site. Enzymatic characterization demonstrated that BcMazG is a nucleoside triphosphate (NTP) pyrophosphohydrolase and prefers adenosine triphosphate as a substrate among canonical NTPs. Moreover, structural comparison of BcMazG with its homologues revealed a potential regulation mechanism whereby the enzymatic activity of BcMazG is regulated by its C-terminal region.

Crystal structure of the Bacillus-conserved MazG protein, a nucleotide pyrophosphohydrolase.

Pyridoxal kinase is a ubiquitous enzyme essential for pyridoxal 5'-phosphate (PLP) homeostasis since PLP is required for the catalytic activity of a variety of PLP-dependent enzymes involved in amino acid, lipid, and sugar metabolism as well as neurotransmitter biosynthesis. Previously, two catalytic mechanisms were proposed with regard to Pdx kinases, in which either the aspartate or the cysteine residue is involved as a catalytic residue. Because the Pdx kinase of Pseudomonas aeruginosa (PaPdxK) contains both residues, the catalytic mechanism of PaPdxK remains elusive. To elucidate the substrate-recognition and catalytic mechanisms of PaPdxK, the crystal structure of PaPdxK was determined at a 2.0 Å resolution. The PaPdxK structure possesses a channel that can accommodate substrates and a metallic cofactor. Our structure-based biochemical and mutational analyses in combination with modeling studies suggest that PaPdxK catalysis is mediated by an acid-base mechanism through the catalytic acid Asp225 and a helical dipole moment.

Crystal structure and catalytic mechanism of pyridoxal kinase from Pseudomonas aeruginosa.

A phenolic acid decarboxylase (padC) regulator, PadR and its homologs proteins belong to the PadR family. Despite the growing numbers of the PadR family members and their various roles in bacteria, such as detoxifications, drug transports and circadian rhythms, biochemical and biophysical studies of the PadR family are very limited. Thus, a ligand-induced regulatory mechanism of the PadR family transcription factors remains to be elucidated. Here, we report a crystal structure of a Bacteroides fragilis PadR-like protein, BF2549 and revealed its interaction with putative operator DNA and ligand molecules. Comparative structural and primary sequence analyses provide a PadR-specific motif that is conserved in the PadR family but deviated from the MarR family. Furthermore, putative ligand binding sites are observed in the BF2549 structure. Finally, a homology-based structure model of BF2549 and 29-mer dsDNA propose regulatory mechanisms of the PadR family in transcriptional derepression.

Structural and functional analysis of BF2549, a PadR-like transcription factor from Bacteroides fragilis.

FliS is a cytoplasmic flagellar chaperone for the flagellin, which polymerizes into filaments outside of the flagellated bacteria. Cytoplasmic interaction between FliS and flagellin is critical to retain the flagellin protein in a monomeric form, which is transported from the cytoplasm through the flagellar export apparatus to the extracellular space for filament assembly. Defects in the FliS protein directly diminish bacterial motility, pathogenicity, and viability. Although the overall structure of FliS is known, structural and mutational studies on FliS from other bacterial species are still required to reveal any unresolved biophysical features of FliS itself or functionally critical residues for flagellin recognition. Here, we present the crystal structure of FliS from Bacillus cereus (BcFliS) at 2.0 Å resolution. FliS possesses a highly dynamic N-terminal region, which is appended to the common four-helix bundle structure. An invariant proline residue (Pro17 in B. cereus FliS) was identified in all known FliS sequences between the N-terminal region and the four-helix bundle. The N-terminal proline residue functions as a helix breaker critical for FliS dimerization and flagellin recognition.

Crystal structure of the flagellar chaperone FliS from Bacillus cereus and an invariant proline critical for FliS dimerization and flagellin recognition.

Meong Il Kim

Division of Biological Science and Technology

Yonsei University

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Meong Il Kim

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Division of Biological Science and Technology

Yonsei University

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Division of Biological Science and Technology