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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: 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.

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

X-ray crystal structure of Pput2725.(A) Chemical structures of 2,4,6-trichlorophenol (TCP) and sphingosine, illustrating the diversity of COG4313 protein transport substrates. (B) Coomassie-stained SDS-PAGE gels showing in vitro folded Pput2725 (left panel) and OM-expressed CnTcpY (right panel). Both proteins are heat-modifiable and migrate at a lower molecular mass without sample boiling because they are still folded (F; -). After boiling (B) the proteins unfold (U) and migrate close to their true molecular mass (30.5 kDa for Pput2725 and 34 kDA for TcpY). (C) Cartoon overview of Pput2725 with rainbow coloring (N-terminus; blue). The right panel is a cut-away view showing the N-terminus inside the barrel lumen. The protein is colored by B-factor (blue; low). The extracellular loops and the N-terminus are labeled. All molecular models were made using Pymol28.
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f1: X-ray crystal structure of Pput2725.(A) Chemical structures of 2,4,6-trichlorophenol (TCP) and sphingosine, illustrating the diversity of COG4313 protein transport substrates. (B) Coomassie-stained SDS-PAGE gels showing in vitro folded Pput2725 (left panel) and OM-expressed CnTcpY (right panel). Both proteins are heat-modifiable and migrate at a lower molecular mass without sample boiling because they are still folded (F; -). After boiling (B) the proteins unfold (U) and migrate close to their true molecular mass (30.5 kDa for Pput2725 and 34 kDA for TcpY). (C) Cartoon overview of Pput2725 with rainbow coloring (N-terminus; blue). The right panel is a cut-away view showing the N-terminus inside the barrel lumen. The protein is colored by B-factor (blue; low). The extracellular loops and the N-terminus are labeled. All molecular models were made using Pymol28.

Mentions: Only two COG4313 family members have been characterised in some detail in the literature. The first is the TcpY protein from Cupriavidis necator (formerly Ralstonia eutropha). This protein is located in the tcp operon which is dedicated to the degradation of trichlorophenol (TCP; Fig. 1A)4. Since TcpY has a signal sequence and is predicted to form a β-barrel structure, Belchik et al. suggested that TcpY could function as an OM uptake channel for TCP. To support this notion it was shown that E. coli transformed with plasmid-encoded tcpY is more susceptible to toxicity caused by TCP in the growth medium, supporting a role for TcpY as an uptake channel4. The second COG4313 protein for which a function has been proposed is the PA5325 gene from the opportunistic pathogen Pseudomonas aeruginosa. PA5325 is strongly upregulated in the presence of sphingosine by the sphingosine-responsive transcription factor sphR (PA5324), and was renamed sphA5. Interestingly, individual deletions of both sphR and sphA result in decreased survival of P. aeruginosa in the murine lung. Sphingosine (Fig. 1A) is a compound with known antimicrobial properties and is a component of lung surfactant. Intriguingly, a sphA deletion results in increased killing of P. aeruginosa in mice. These data are consistent with a role for SphA as an uptake channel for sphingosine to protect the cell from the harmful affects of this surfactant, possibly by metabolising it5. With regards to the channel studied here, encoded by orf Pput2725 from the biodegrader Pseudomonas putida F1 (PpF1), no function has yet been established. However, the neighboring gene Pput2724 as well as Pput2727-2729 code for enzymes with roles in the degradation of monoaromatic hydrocarbons (MAH), suggesting a possible role for the Pput2725 protein in OM MAH uptake. Pput2725 is the only COG4313 family member in PpF1.


Crystal structure of a COG4313 outer membrane channel.

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

X-ray crystal structure of Pput2725.(A) Chemical structures of 2,4,6-trichlorophenol (TCP) and sphingosine, illustrating the diversity of COG4313 protein transport substrates. (B) Coomassie-stained SDS-PAGE gels showing in vitro folded Pput2725 (left panel) and OM-expressed CnTcpY (right panel). Both proteins are heat-modifiable and migrate at a lower molecular mass without sample boiling because they are still folded (F; -). After boiling (B) the proteins unfold (U) and migrate close to their true molecular mass (30.5 kDa for Pput2725 and 34 kDA for TcpY). (C) Cartoon overview of Pput2725 with rainbow coloring (N-terminus; blue). The right panel is a cut-away view showing the N-terminus inside the barrel lumen. The protein is colored by B-factor (blue; low). The extracellular loops and the N-terminus are labeled. All molecular models were made using Pymol28.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: X-ray crystal structure of Pput2725.(A) Chemical structures of 2,4,6-trichlorophenol (TCP) and sphingosine, illustrating the diversity of COG4313 protein transport substrates. (B) Coomassie-stained SDS-PAGE gels showing in vitro folded Pput2725 (left panel) and OM-expressed CnTcpY (right panel). Both proteins are heat-modifiable and migrate at a lower molecular mass without sample boiling because they are still folded (F; -). After boiling (B) the proteins unfold (U) and migrate close to their true molecular mass (30.5 kDa for Pput2725 and 34 kDA for TcpY). (C) Cartoon overview of Pput2725 with rainbow coloring (N-terminus; blue). The right panel is a cut-away view showing the N-terminus inside the barrel lumen. The protein is colored by B-factor (blue; low). The extracellular loops and the N-terminus are labeled. All molecular models were made using Pymol28.
Mentions: Only two COG4313 family members have been characterised in some detail in the literature. The first is the TcpY protein from Cupriavidis necator (formerly Ralstonia eutropha). This protein is located in the tcp operon which is dedicated to the degradation of trichlorophenol (TCP; Fig. 1A)4. Since TcpY has a signal sequence and is predicted to form a β-barrel structure, Belchik et al. suggested that TcpY could function as an OM uptake channel for TCP. To support this notion it was shown that E. coli transformed with plasmid-encoded tcpY is more susceptible to toxicity caused by TCP in the growth medium, supporting a role for TcpY as an uptake channel4. The second COG4313 protein for which a function has been proposed is the PA5325 gene from the opportunistic pathogen Pseudomonas aeruginosa. PA5325 is strongly upregulated in the presence of sphingosine by the sphingosine-responsive transcription factor sphR (PA5324), and was renamed sphA5. Interestingly, individual deletions of both sphR and sphA result in decreased survival of P. aeruginosa in the murine lung. Sphingosine (Fig. 1A) is a compound with known antimicrobial properties and is a component of lung surfactant. Intriguingly, a sphA deletion results in increased killing of P. aeruginosa in mice. These data are consistent with a role for SphA as an uptake channel for sphingosine to protect the cell from the harmful affects of this surfactant, possibly by metabolising it5. With regards to the channel studied here, encoded by orf Pput2725 from the biodegrader Pseudomonas putida F1 (PpF1), no function has yet been established. However, the neighboring gene Pput2724 as well as Pput2727-2729 code for enzymes with roles in the degradation of monoaromatic hydrocarbons (MAH), suggesting a possible role for the Pput2725 protein in OM MAH uptake. Pput2725 is the only COG4313 family member in PpF1.

Bottom Line: 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.

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