TY - JOUR
T1 - Expression, purification and crystallization of the phosphate-binding PstS protein from Pseudomonas aeruginosa
AU - Neznansky, Avi
AU - Opatowsky, Yarden
PY - 2014/7
Y1 - 2014/7
N2 - Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C2221, with unit-cell parameters a = 67.5, b = 151.3, c = 108.9Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.09 Å3 Da-1 and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 35.4, b = 148.3, c = 216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.14 Å3 Da-1 and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.
AB - Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune-suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate-transport system of PA, plays a critical role in the establishment of biofilms. In some drug-resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour-diffusion method at 20°C and X-ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C2221, with unit-cell parameters a = 67.5, b = 151.3, c = 108.9Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.09 Å3 Da-1 and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P212121, with unit-cell parameters a = 35.4, b = 148.3, c = 216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight (V M) of 2.14 Å3 Da-1 and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P212121 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.
KW - Pseudomonas aeruginosa
KW - PstS
UR - http://www.scopus.com/inward/record.url?scp=84905513582&partnerID=8YFLogxK
U2 - 10.1107/S2053230X14010279
DO - 10.1107/S2053230X14010279
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C2 - 25005086
AN - SCOPUS:84905513582
SN - 1744-3091
VL - 70
SP - 906
EP - 910
JO - Acta Crystallographica Section F:Structural Biology Communications
JF - Acta Crystallographica Section F:Structural Biology Communications
IS - 7
ER -