Preliminary time-of-flight neutron diffraction studies ofEscherichia coliABC transport receptor phosphate-binding protein at the Protein Crystallography Station

Inorganic phosphate is an essential molecule for all known life. Organisms have developed many mechanisms to ensure an adequate supply, even in low-phosphate conditions. In prokaryotes phosphate transport is instigated by the phosphate-binding protein (PBP), the initial receptor for the ATP-binding cassette (ABC) phosphate transporter. In the crystal structure of the PBP–phosphate complex, the phosphate is completely desolvated and sequestered in a deep cleft and is bound by 13 hydrogen bonds: 12 to protein NH and OH donor groups and one to a carboxylate acceptor group. The carboxylate plays a key recognition role by accepting a phosphate hydrogen. PBP phosphate affinity is relatively consistent across a broad pH range, indicating the capacity to bind monobasic (H2PO4−) and dibasic (HPO42−) phosphate; however, the mechanism by which it might accommodate the second hydrogen of monobasic phosphate is unclear. To answer this question, neutron diffraction studies were initiated. Large single crystals with a volume of 8 mm3were grown and subjected to hydrogen/deuterium exchange. A 2.5 Å resolution data set was collected on the Protein Crystallography Station at the Los Alamos Neutron Science Center. Initial refinement of the neutron data shows significant nuclear density, and refinement is ongoing. This is the first report of a neutron study from this superfamily.

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The high-affinity phosphate-binding protein PstS is accumulated under high fructose concentrations and mutation of the corresponding gene affects differentiation in Streptomyces lividans

Microbiology ◽

10.1099/mic.0.27983-0 ◽

2005 ◽

Vol 151 (8) ◽

pp. 2583-2592 ◽

Cited By ~ 42

Author(s):

Margarita Díaz ◽

Ana Esteban ◽

José Manuel Fernández-Abalos ◽

Ramón I. Santamaría

Keyword(s):

Culture Media ◽

Binding Protein ◽

Protein Pattern ◽

Phosphate Transport ◽

Streptomyces Lividans ◽

Null Mutant ◽

Phosphate Binding ◽

Genetic Switch ◽

High Affinity ◽

Phosphate Binding Protein

The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Δppk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.

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Studies of phosphate transport in Escherichia Coli. I. Reexamination of the effect of osmotic and cold shock on phosphate uptake and some attempts to restore uptake with phosphate binding protein

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(76)90281-9 ◽

1976 ◽

Vol 433 (3) ◽

pp. 555-563 ◽

Cited By ~ 7

Author(s):

A.S. Rae ◽

K.P. Strickland ◽

N. Medveczky ◽

H. Rosenberg

Keyword(s):

Escherichia Coli ◽

Cold Shock ◽

Phosphate Uptake ◽

Binding Protein ◽

Phosphate Transport ◽

Phosphate Binding ◽

Phosphate Binding Protein

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Restoration of phosphate transport by the phosphate-binding protein in spheroplasts of Escherichia coli.

Journal of Bacteriology ◽

10.1128/jb.131.2.512-518.1977 ◽

1977 ◽

Vol 131 (2) ◽

pp. 512-518 ◽

Cited By ~ 27

Author(s):

R G Gerdes ◽

K P Strickland ◽

H Rosenberg

Keyword(s):

Escherichia Coli ◽

Binding Protein ◽

Phosphate Transport ◽

Phosphate Binding ◽

Phosphate Binding Protein

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The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316010433 ◽

2016 ◽

Vol 72 (8) ◽

pp. 933-943 ◽

Cited By ~ 11

Author(s):

Ronan Keegan ◽

David G. Waterman ◽

David J. Hopper ◽

Leighton Coates ◽

Graham Taylor ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Binding Protein ◽

Data Bank ◽

Crystal Form ◽

Atomic Resolution ◽

Search Model ◽

Molecular Replacement ◽

Phosphate Binding ◽

Data Set ◽

Phosphate Binding Protein

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) fromAlcaligenessp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement usingMOLREPin which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in theMOLREPpeak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. ABLASTsearch then indicated that the molecule was most probably a phosphate-binding protein fromStenotrophomonas maltophilia(UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of theS. maltophiliaprotein has been refined to anRfactor of 10.15% and anRfreeof 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.

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