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Crystal structure of a COG4313 outer membrane channel.

van den Berg B, Bhamidimarri SP, Winterhalter M - Sci Rep (2015)

Bottom Line: Structure-function studies of this protein family have so far been hampered by a lack of structural information.Instead, the presence of bound detergent molecules inside the barrel suggests that Pput2725 mediates uptake of hydrophobic molecules.Sequence alignments and the locations of highly conserved residues suggest the presence of a dynamic lateral opening through which hydrophobic molecules might gain entry into the cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

ABSTRACT
COG4313 proteins form a large and widespread family of outer membrane channels and have been implicated in the uptake of a variety of hydrophobic molecules. Structure-function studies of this protein family have so far been hampered by a lack of structural information. Here we present the X-ray crystal structure of Pput2725 from the biodegrader Pseudomonas putida F1, a COG4313 channel of unknown function, using data to 2.3 Å resolution. The structure shows a 12-stranded barrel with an N-terminal segment preceding the first β-strand occluding the lumen of the barrel. Single channel electrophysiology and liposome swelling experiments suggest that while the narrow channel visible in the crystal structure does allow passage of ions and certain small molecules in vitro, Pput2725 is unlikely to function as a channel for hydrophilic molecules. Instead, the presence of bound detergent molecules inside the barrel suggests that Pput2725 mediates uptake of hydrophobic molecules. Sequence alignments and the locations of highly conserved residues suggest the presence of a dynamic lateral opening through which hydrophobic molecules might gain entry into the cell. Our results provide the basis for structure-function studies of COG4313 family members with known function, such as the SphA sphingosine uptake channel of Pseudomonas aeruginosa.

No MeSH data available.


Related in: MedlinePlus

ClustalW sequence alignment of the COG4313 channels Pput2725, C. necator TcpY and P. aeruginosa SphA.The signal sequences as predicted by SignalP are shown in green. The observed secondary structure elements based on the crystal structure of Pput2725 are indicated (β-strands; blue, helices; red). Identical residues (*) located within the putative lateral exit site are highlighted in pink.
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f5: ClustalW sequence alignment of the COG4313 channels Pput2725, C. necator TcpY and P. aeruginosa SphA.The signal sequences as predicted by SignalP are shown in green. The observed secondary structure elements based on the crystal structure of Pput2725 are indicated (β-strands; blue, helices; red). Identical residues (*) located within the putative lateral exit site are highlighted in pink.

Mentions: To obtain more information regarding function we performed a sequence alignment of Pput2725 with C. necator TcpY (CnTcpY) and P. aeruginosa SphA (PaSphA), the two COG4313 family members for which a function has been described. As is typical for the entire family, pairwise sequence identities between the three proteins are low (typically less than 20%), which should increase the information content of highly conserved residues. For the three COG4313 proteins analysed, there are only seventeen identical residues (Fig. 5). Two of these residues (Glu2 and Gly6) are located in the N-terminus. The side chain of Glu2 interacts with the side chain of Asn225 in the barrel wall, stabilising the N-terminus. Gly6 might be important for folding of the N-terminus in the confined lumen of the barrel. Interestingly, the alignment also suggests that the N-termini (i.e. the segments preceding the first β-strand) of C. necator TcpY and P. aeruginosa SphA are approximately twice the length compared to that of Pput2725 (Fig. 5). Assuming that these longer N-termini also occlude the lumen of the TcpY and SphA barrels this suggests that these proteins may not have the narrow channel that is present in Pput2725. This reinforces the notion from the transport experiments described above that COG4313 proteins likely do not function as classical uptake channels for hydrophilic molecules.


Crystal structure of a COG4313 outer membrane channel.

van den Berg B, Bhamidimarri SP, Winterhalter M - Sci Rep (2015)

ClustalW sequence alignment of the COG4313 channels Pput2725, C. necator TcpY and P. aeruginosa SphA.The signal sequences as predicted by SignalP are shown in green. The observed secondary structure elements based on the crystal structure of Pput2725 are indicated (β-strands; blue, helices; red). Identical residues (*) located within the putative lateral exit site are highlighted in pink.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493636&req=5

f5: ClustalW sequence alignment of the COG4313 channels Pput2725, C. necator TcpY and P. aeruginosa SphA.The signal sequences as predicted by SignalP are shown in green. The observed secondary structure elements based on the crystal structure of Pput2725 are indicated (β-strands; blue, helices; red). Identical residues (*) located within the putative lateral exit site are highlighted in pink.
Mentions: To obtain more information regarding function we performed a sequence alignment of Pput2725 with C. necator TcpY (CnTcpY) and P. aeruginosa SphA (PaSphA), the two COG4313 family members for which a function has been described. As is typical for the entire family, pairwise sequence identities between the three proteins are low (typically less than 20%), which should increase the information content of highly conserved residues. For the three COG4313 proteins analysed, there are only seventeen identical residues (Fig. 5). Two of these residues (Glu2 and Gly6) are located in the N-terminus. The side chain of Glu2 interacts with the side chain of Asn225 in the barrel wall, stabilising the N-terminus. Gly6 might be important for folding of the N-terminus in the confined lumen of the barrel. Interestingly, the alignment also suggests that the N-termini (i.e. the segments preceding the first β-strand) of C. necator TcpY and P. aeruginosa SphA are approximately twice the length compared to that of Pput2725 (Fig. 5). Assuming that these longer N-termini also occlude the lumen of the TcpY and SphA barrels this suggests that these proteins may not have the narrow channel that is present in Pput2725. This reinforces the notion from the transport experiments described above that COG4313 proteins likely do not function as classical uptake channels for hydrophilic molecules.

Bottom Line: Structure-function studies of this protein family have so far been hampered by a lack of structural information.Instead, the presence of bound detergent molecules inside the barrel suggests that Pput2725 mediates uptake of hydrophobic molecules.Sequence alignments and the locations of highly conserved residues suggest the presence of a dynamic lateral opening through which hydrophobic molecules might gain entry into the cell.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

ABSTRACT
COG4313 proteins form a large and widespread family of outer membrane channels and have been implicated in the uptake of a variety of hydrophobic molecules. Structure-function studies of this protein family have so far been hampered by a lack of structural information. Here we present the X-ray crystal structure of Pput2725 from the biodegrader Pseudomonas putida F1, a COG4313 channel of unknown function, using data to 2.3 Å resolution. The structure shows a 12-stranded barrel with an N-terminal segment preceding the first β-strand occluding the lumen of the barrel. Single channel electrophysiology and liposome swelling experiments suggest that while the narrow channel visible in the crystal structure does allow passage of ions and certain small molecules in vitro, Pput2725 is unlikely to function as a channel for hydrophilic molecules. Instead, the presence of bound detergent molecules inside the barrel suggests that Pput2725 mediates uptake of hydrophobic molecules. Sequence alignments and the locations of highly conserved residues suggest the presence of a dynamic lateral opening through which hydrophobic molecules might gain entry into the cell. Our results provide the basis for structure-function studies of COG4313 family members with known function, such as the SphA sphingosine uptake channel of Pseudomonas aeruginosa.

No MeSH data available.


Related in: MedlinePlus