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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

The N-terminus constricts the barrel lumen of Pput2725.(A) Views from the extracellular side showing a cartoon (top panel; colored by B-factor) and a molecular surface model (bottom panel). (B) Cut-away surface view showing the internal channel. The constriction is indicated with an arrow. (C) Surface view from the extracellular side of Pput2725 lacking the N-terminus, showing a large channel. For comparison, a rainbow surface model is shown for E. coli NanC, which is involved in the uptake of N-acetylneuraminic acid.
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f2: The N-terminus constricts the barrel lumen of Pput2725.(A) Views from the extracellular side showing a cartoon (top panel; colored by B-factor) and a molecular surface model (bottom panel). (B) Cut-away surface view showing the internal channel. The constriction is indicated with an arrow. (C) Surface view from the extracellular side of Pput2725 lacking the N-terminus, showing a large channel. For comparison, a rainbow surface model is shown for E. coli NanC, which is involved in the uptake of N-acetylneuraminic acid.

Mentions: The crystal structure for the Pput2725 channel reveals a monomeric 12-stranded β-barrel, confirming the topology predictions for this family member and demonstrating that contrary to a previous notion made for TcpY4, COG4313 proteins do not have the same topology as FadL channels, which have 14-stranded barrels. Electron density for Pput2725 is visible for the entire polypeptide, with the exception of a number of exposed side chains that are presumably disordered. On the extracellular side, there are no loops that fold inwards to constrict the channel as observed for barrels with larger diameters. However, on the periplasmic side the N-terminal fourteen residues occupy the lumen of the barrel (Fig. 1C). This is a marked contrast with the structures of members of the KdgM family, which are so far the only other 12-stranded uptake channels for which structures have been solved1112. For example, the structure of the E. coli N-Acetylneuraminic acid channel NanC shows a large channel of uniform diameter that is not constricted by the N-terminus (Fig. 2C). Thus, the barrel-inserted N-terminus of Pput2725 is a novel feature for 12-stranded OM uptake channels. The Pput2725 structure resembles that of the post-cleavage state of autotransporter proteins, which are not uptake channels for small molecules but protein secretion devices13. The structural similarity of Pput2725 to autotransporters is confirmed by a DALI search14, which shows that the most similar proteins to Pput2725 are all β-barrel domains of autotransporters, with the serine protease EspP being the top hit (PDB ID 3SLT; Z-score 22, 2.6 Å r.m.s.d.). It should be noted that while these proteins are similar in structure, the r.m.s.d. values are still relatively large and rationalise the failure of molecular replacement trials in solving the structure of Pput2725.


Crystal structure of a COG4313 outer membrane channel.

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

The N-terminus constricts the barrel lumen of Pput2725.(A) Views from the extracellular side showing a cartoon (top panel; colored by B-factor) and a molecular surface model (bottom panel). (B) Cut-away surface view showing the internal channel. The constriction is indicated with an arrow. (C) Surface view from the extracellular side of Pput2725 lacking the N-terminus, showing a large channel. For comparison, a rainbow surface model is shown for E. coli NanC, which is involved in the uptake of N-acetylneuraminic acid.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The N-terminus constricts the barrel lumen of Pput2725.(A) Views from the extracellular side showing a cartoon (top panel; colored by B-factor) and a molecular surface model (bottom panel). (B) Cut-away surface view showing the internal channel. The constriction is indicated with an arrow. (C) Surface view from the extracellular side of Pput2725 lacking the N-terminus, showing a large channel. For comparison, a rainbow surface model is shown for E. coli NanC, which is involved in the uptake of N-acetylneuraminic acid.
Mentions: The crystal structure for the Pput2725 channel reveals a monomeric 12-stranded β-barrel, confirming the topology predictions for this family member and demonstrating that contrary to a previous notion made for TcpY4, COG4313 proteins do not have the same topology as FadL channels, which have 14-stranded barrels. Electron density for Pput2725 is visible for the entire polypeptide, with the exception of a number of exposed side chains that are presumably disordered. On the extracellular side, there are no loops that fold inwards to constrict the channel as observed for barrels with larger diameters. However, on the periplasmic side the N-terminal fourteen residues occupy the lumen of the barrel (Fig. 1C). This is a marked contrast with the structures of members of the KdgM family, which are so far the only other 12-stranded uptake channels for which structures have been solved1112. For example, the structure of the E. coli N-Acetylneuraminic acid channel NanC shows a large channel of uniform diameter that is not constricted by the N-terminus (Fig. 2C). Thus, the barrel-inserted N-terminus of Pput2725 is a novel feature for 12-stranded OM uptake channels. The Pput2725 structure resembles that of the post-cleavage state of autotransporter proteins, which are not uptake channels for small molecules but protein secretion devices13. The structural similarity of Pput2725 to autotransporters is confirmed by a DALI search14, which shows that the most similar proteins to Pput2725 are all β-barrel domains of autotransporters, with the serine protease EspP being the top hit (PDB ID 3SLT; Z-score 22, 2.6 Å r.m.s.d.). It should be noted that while these proteins are similar in structure, the r.m.s.d. values are still relatively large and rationalise the failure of molecular replacement trials in solving the structure of Pput2725.

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