We have determined the crystal structure of the GDP complex of the YjeQ protein from Thermotoga maritima (TmYjeQ), a member of the YjeQ GTPase subfamaily. TmYjeQ, a homologue of Escherichia coli YjeQ, which is known to bind to the ribosome, is composed of three domains: an N

PhoU proteins are known to play a role in the regulation of phosphate uptake. In Thermotoga maritima, two PhoU homologues have been identified bioinformatically. Here we report the crystal structure of one of the PhoU homologues at 2.0 Å resolution. The structure of the PhoU protein homologue contains a highly symmetric new structural fold composed of two repeats of a three-helix bundle. The structure unexpectedly revealed a trinuclear and a tetranuclear iron cluster that were found to be bound on the surface. Each of the two multinuclear iron clusters is coordinated by a conserved E(D)XXXD motif pair. Our structure reveals a new class of metalloprotein containing multinuclear iron clusters. The possible functional implication based on the structure are discussed.Inorganic phosphate (P i ) uptake is of fundamental importance in the cell physiology of bacteria because P i is required as a nutrient. Escherichia coli has developed a P i acquisition system that allows the assimilation of P i via a variety of systems. Two distinct systems for the uptake of P i have been described: the low affinity phosphate inorganic transporter and the high affinity phosphate-specific transporter (PstSCAB) (1, 2). When the preferred P i source is in excess, it is taken up by the phosphate inorganic transporter. When the extracellular P i concentration is less than ϳ4 M, the synthesis of the high affinity transporter is induced, and P i is taken up by PstSCAB. The PstSCAB transporter belongs to the superfamily of ATPbinding cassette transporters and is encoded by the pst operon (3, 4). This operon contains five genes that are transcribed counterclockwise in the following order: pstS, pstC, pstA, pstB, and phoU. PstS is a periplasmic P i -binding protein, and PstC and PstA are integral membrane proteins that mediate the translocation of P i through the inner membrane. PstB is an ATPase that energizes the transport. The Pst operon is part of the phosphate (PHO) 1 regulon that consists of 31 genes arranged in eight different operons in E. coli (5, 6). The genes and operons of the PHO regulon are co-regulated by a two-component system composed of the regulatory proteins PhoB and PhoR. When the concentration of P i in the medium falls below ϳ4 M, the sensor protein PhoR phosphorylates PhoB, and the phosphorylated PhoB binds to the PHO boxes in the control region of Pst, recruiting the 70 subunit of the RNA polymerase and initiating transcription. When the P i concentration in the medium is in excess, the PHO regulon is repressed. Repression of the PHO regulon requires not only an excess concentration of extracellular P i but also the intact PstSCAB transporter and the PhoU protein.The PhoU gene encodes a polypeptide of molecular mass ϳ27,000 Da. Although it is located in the pst operon, the encoded PhoU protein does not seem to participate in P i transport (4). Besides its role as a repressor of the PHO regulon, PhoU was also reported to be involved in intracellular P i metabolism (presumably related to the synthesis of ATP) (7).Currently...

Crystal structures of the bacterial multidrug transporter AcrB in R32 and C2 space groups showing both symmetric and asymmetric trimeric assemblies respectively, supplemented with biochemical investigations, have provided most of the structural basis for a molecular level understanding of the protein structure and mechanisms for substrate uptake and translocation carried out by this 114 kDa inner membrane protein. They suggest that AcrB captures ligands primarily from the periplasm. Substrates can also enter the inner cavity of the transporter from the cytoplasm, but the exact mechanism of this remains undefined.Address correspondence to: Prof. Sung-Hou Kim, shkim@cchem.berkeley.edu or Debanu Das, ddas@lbl.gov. * These authors contributed equally to the work Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public AccessAuthor Manuscript J Struct Biol. Author manuscript; available in PMC 2007 October 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAnalysis of the amino acids sequences of AcrB and its homologs revealed the presence of conserved residues at the N-terminus including two phenylalanines which may be exposed to the cytoplasm. Any potential role that these conserved residues may play in function has not been addressed by existing biochemical or structural studies. Since phenylalanine residues elsewhere in the protein have been implicated in ligand binding, we explored the structure of this N-terminal region to investigate structural determinants near the cytoplasmic opening that may mediate drug uptake. Our structure of AcrB in R32 space group reveals an N-terminus loop, reducing the diameter of the central opening to ∼15 Å as opposed to the previously reported value of ∼30 Å for crystal structures in this space group with disordered N-terminus. Recent structures of the AcrB in C2 space group have revealed a helical conformation of this N-terminus but have not discussed its possible implications. We present the crystal structure of AcrB that reveals the structure of the N-terminus containing the conserved residues. We hope that the structural information provides a structural basis for others to design further biochemical investigation of the role of this portion of AcrB in mediating cytoplasmic ligand discrimination and uptake.

NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.