Genome analyses have revealed that members of the Lrp/AsnC family of transcriptional regulators are widely distributed among prokaryotes, including both bacteria and archaea. These regulatory proteins are involved in cellular metabolism in both global and specific manners, depending on the availability of the exogenous amino acid effectors. Here we report the first crystal structure of glutamine receptor protein (Grp) from Sulfolobus tokodaii strain 7, in the ligand-free and glutamine-bound (Grp-Gln) forms. Although the overall structures of both molecules are similar, a significant conformational change was observed at the ligand [L-glutamine (Gln)] binding site in the effector domain, which may be essential for further stabilization of the octameric structure, and in turn for facilitating DNA binding. In addition, we predicted promoter for the grp gene, and these analyses suggested the importance of cooperative binding to the protein. To gain insights into the ligand-induced conformational changes, we mutated all of the ligand-binding residues in Grp, and revealed the importance of Gln binding by biochemical and structural analyses. Further structural analyses showed that Y77 is crucial for ligand binding, and that the residues T132 and T134, which are highly conserved among the Lrp family of proteins, fluctuates between the active and inactive conformations, thus affecting protein oligomerization for DNA binding.

Crystal structure of glutamine receptor protein from Sulfolobus tokodaii strain 7 in complex with its effector L-glutamine: implications of effector binding in molecular association and DNA binding

Genome-scale regulatory networks are a great concern in studying the crucial problems in bacterial genome biology. Methanothermobacter thermautotrophicus ΔH (MTH) is a model organism of thermophilic methanogenic archaea contributing to a large environmental impact in global carbon cycling and strain used in the biofuel industry. Nonetheless, systematic transcriptional regulations have not been previously made for this organism. In the present study, a global regulatory network of this organism was reconstructed and its associated molecular function revealed at systems-level. The inferred genome-wide regulatory network comprises 25 predicted regulons with 1168 associating genes constructed from the previously characterized and uncharacterized transcription factors. Regulatory networks qualitatively assessed with gene ontology terms for functional confidence, whereas network robustness validated with topological parameters. We predicted rhlR (164 genes), cspA (132 genes), algU (141 genes), gcvA (26 genes) and lexA (97 genes) regulons. The predicted regulons mainly contributed to the transcriptional regulation of carbohydrate metabolism, stress response, metal homeostasis, and amino acid biosynthesis. Six DNA binding motifs predicted from MTH independently associated with more than 100 genes, reflecting that gene regulation has been controlled directly or indirectly under the respective promoter site. The results of this study substantially expanded the existing knowledge of its transcriptional regulatory mechanisms. The regulatory network model can be used for genetic experiments, functional annotations of genes, metabolic reconstruction and evolutionary analysis.

Modeling a global regulatory network of Methanothermobacter thermautotrophicus strain ∆H

Rv2779c from Mycobacterium tuberculosis is a feast/famine regulatory protein. This class of proteins are also known as the leucine-responsive regulatory protein/asparagine synthase C family (Lrp/AsnC) of transcriptional regulators and are known to be involved in various metabolic processes in bacteria and fungi. They contain a RAM (regulator of amino-acid metabolism) domain that is rarely found in humans and acts as the oligomerization domain. Since the oligomeric status is often linked to the particular functional role in these proteins, binding of ligands to the domain can elicit specific functional responses. Full-length Rv2779c corresponding to a molecular mass of 19.8 kDa and 179 residues was cloned and purified to homogeneity following transformation into Escherichia coli C41 (DE3) cells. Crystals were grown by vapour diffusion using the hanging-drop method. Diffraction data extending to 2.8 A resolution were collected from a single crystal that belonged to space group P21212, with unit-cell parameters a = 99.6, b = 146.0, c = 49.9 A. Matthews coefficient (VM) calculations suggest that four molecules are present in the asymmetric unit, corresponding to a solvent content of ∼46%. Molecular-replacement calculations using the crystal structure of a homologue, Rv3291c, as the search model gave an unambiguous solution corresponding to four subunits in the asymmetric unit.

/pdf/cloning-overexpression-purification-and-preliminary-x-ray-41jlmxlhp8.pdf

Cloning, overexpression, purification and preliminary X-ray analysis of a feast/famine regulatory protein (Rv2779c) from Mycobacterium tuberculosis H37Rv

Interactions between the archaeal transcription repressor FL11 and its coregulators lysine and arginine Mitsugu Yamada, Sanae A. Ishijima, and Masashi Suzuki* 1 Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Sanbancho 5, Chiyoda-ku, Tokyo 102-0075, Japan 2 National Institute of Advanced Industrial Science and Technology, Tsukuba Center 6-10, Higashi 1-1-1, Tsukuba 305-8566, Japan

https://onlinelibrary.wiley.com/doi/pdf/10.1002/prot.22269

Interactions between the archaeal transcription repressor FL11 and its coregulators lysine and arginine.

Rv3291c (MtbLrpA), a transcriptional regulator, belongs to the leucine-responsive regulatory protein (Lrp) family and is thought to play an important role in Mycobacterium tuberculosis persistence. In this study, we verified 17 novel potential binding sites for MtbLrpA by in vitro binding assays on the basis of previous predictions from an in silico analysis and bacterial one-hybrid (BIH) reporter system. Amino acids, such as tyrosine, phenylalanine, tryptophan, and histidine, strongly affect the binding affinity of MtbLrpA, and vitamins, including B1, B3, B6, VC, B7, B9, B12, VA, and VK3, also decrease MtbLrpA binding affinity. This is the first report regarding that an Lrp-like protein can sense vitamins as an environmental signal. Vitamin supplementation to the environment can change the expression level of the target genes, which provides a potential mechanism for tuberculosis supplementary treatment with vitamins.

New Targets and Cofactors for the Transcription Factor LrpA from Mycobacterium tuberculosis.

The putative GTPase PH0525 from P. horikoshii OT3 was crystallized using the microbatch method. Crystals were formed under two different conditions, providing two distinct crystal forms. Diffraction data from the two forms were measured to resolution limits of 2.30 and 2.40 A and processed in space groups P2{sub 1} and C222{sub 1}, respectively. GTPases are involved in diverse cellular functions including cell proliferation, cytoskeleton organization and intracellular traffic. The putative GTPase PH0525 from Pyrococcus horikoshii OT3 has been overexpressed in Escherichia coli and purified. Two distinct crystal forms were grown by the microbatch method at 291 K using a very high protein concentration (80 mg ml{sup −1}). Native data sets extending to resolutions of 2.3 and 2.4 A have been collected and processed in space groups P2{sub 1} and C222{sub 1}, respectively. Assuming the presence of one monomer per asymmetric unit gives V{sub M} values of 2.6 and 2.4 A{sup 3} Da{sup −1} for the P2{sub 1} and C222{sub 1} forms, respectively, which is consistent with dynamic light-scattering experiments, which show a monomeric state of the protein in solution.

Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3

The Lrp/AsnC family of transcriptional regulators, also known as feast/famine transcriptional regulators, are widely distributed among bacteria and archaea. This family of proteins are likely to be involved in cellular metabolism, with exogenous amino acids functioning as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of ST1022, a member of the Lrp/AsnC family of proteins, is reported with and without exogenous glutamine as the effector molecule. The crystals of native ST1022 and of the putative complex belong to the tetragonal space group I422, with unit-cell parameters a = b = 103.771, c = 73.297 A and a = b = 103.846, c = 73.992 A, respectively. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of one monomer per asymmetric unit.

Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

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Purification, crystallization and initial X-ray crystallographic analysis of the putative GTPase PH0525 from Pyrococcus horikoshii OT3

Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7