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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

Ion currents and small molecule transport mediated by Pput2725.(A) Ion current trace of three subsequent single channel insertions into a solvent free membrane of DPhPC at +250 mV applied voltage, each giving rise to a single conductance state of ~30 pA. The lower panel shows the ion current amplitude histogram of this trace, indicating in addition to the zero conductive state a distribution around three distinctive current states. The conductance value of each channel is ~126 pS. Experimental conditions are 1M KCl 10 mM MES pH 6 and T = 20 °C. (B) Current (I) vs. Voltage (V) curve of Pput2725. The I–V curve is obtained from at least four independent measurements. (C) Relative substrate uptake rates shown as ΔOD400 values for the large-diameter OmpF porin from E. coli and for Pput2725. The following substrates were tested for Pput2725: glycine (G), glutamic acid (E), glutamine (Q), lysine (K), glucose and sucrose. Representative averages for 3–4 experiments are shown together with their standard deviations.
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f3: Ion currents and small molecule transport mediated by Pput2725.(A) Ion current trace of three subsequent single channel insertions into a solvent free membrane of DPhPC at +250 mV applied voltage, each giving rise to a single conductance state of ~30 pA. The lower panel shows the ion current amplitude histogram of this trace, indicating in addition to the zero conductive state a distribution around three distinctive current states. The conductance value of each channel is ~126 pS. Experimental conditions are 1M KCl 10 mM MES pH 6 and T = 20 °C. (B) Current (I) vs. Voltage (V) curve of Pput2725. The I–V curve is obtained from at least four independent measurements. (C) Relative substrate uptake rates shown as ΔOD400 values for the large-diameter OmpF porin from E. coli and for Pput2725. The following substrates were tested for Pput2725: glycine (G), glutamic acid (E), glutamine (Q), lysine (K), glucose and sucrose. Representative averages for 3–4 experiments are shown together with their standard deviations.

Mentions: Upon reconstitution into a planar lipid bilayer, Pput2725 forms narrow channels with an average conductance of 126 pS in 1 M KCl 10 mM MES at pH6 (Fig. 3A). Surprisingly however, open channels are observed only at voltages higher than ±150 mV; at lower voltages the channels are predominantly in a non-conducting state. Given that any potential difference across the OM will be very small if at all present, the electrophysiology data suggest that Pput2725 may not form an ion-conducting channel under physiological conditions. The relatively low conductance values suggest that even at high voltages the N-terminus may be present inside the barrel lumen, as observed in the crystal structure. By comparison, the single-channel conductance observed for NanC in the presence of 1 M KCl (~350 pS at pH 7) is almost 3-fold higher than Pput2725, and the NanC traces are also quite stable15. The ion current traces for Pput2725 are very noisy and indicate a fluctuating behavior of the narrow channel that likely reflects movement of the N-terminus within the interior of the barrel. The channel is slightly more open in terms of conductance and duration of the open state at positive voltages compared to negative voltages. At high voltages the ionic current through the channel increases linearly with applied voltage (Fig. 3B). Together the electrophysiology data indicate the presence of narrow channel in Pput2725 that requires a substantial input of energy in order to open.


Crystal structure of a COG4313 outer membrane channel.

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

Ion currents and small molecule transport mediated by Pput2725.(A) Ion current trace of three subsequent single channel insertions into a solvent free membrane of DPhPC at +250 mV applied voltage, each giving rise to a single conductance state of ~30 pA. The lower panel shows the ion current amplitude histogram of this trace, indicating in addition to the zero conductive state a distribution around three distinctive current states. The conductance value of each channel is ~126 pS. Experimental conditions are 1M KCl 10 mM MES pH 6 and T = 20 °C. (B) Current (I) vs. Voltage (V) curve of Pput2725. The I–V curve is obtained from at least four independent measurements. (C) Relative substrate uptake rates shown as ΔOD400 values for the large-diameter OmpF porin from E. coli and for Pput2725. The following substrates were tested for Pput2725: glycine (G), glutamic acid (E), glutamine (Q), lysine (K), glucose and sucrose. Representative averages for 3–4 experiments are shown together with their standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Ion currents and small molecule transport mediated by Pput2725.(A) Ion current trace of three subsequent single channel insertions into a solvent free membrane of DPhPC at +250 mV applied voltage, each giving rise to a single conductance state of ~30 pA. The lower panel shows the ion current amplitude histogram of this trace, indicating in addition to the zero conductive state a distribution around three distinctive current states. The conductance value of each channel is ~126 pS. Experimental conditions are 1M KCl 10 mM MES pH 6 and T = 20 °C. (B) Current (I) vs. Voltage (V) curve of Pput2725. The I–V curve is obtained from at least four independent measurements. (C) Relative substrate uptake rates shown as ΔOD400 values for the large-diameter OmpF porin from E. coli and for Pput2725. The following substrates were tested for Pput2725: glycine (G), glutamic acid (E), glutamine (Q), lysine (K), glucose and sucrose. Representative averages for 3–4 experiments are shown together with their standard deviations.
Mentions: Upon reconstitution into a planar lipid bilayer, Pput2725 forms narrow channels with an average conductance of 126 pS in 1 M KCl 10 mM MES at pH6 (Fig. 3A). Surprisingly however, open channels are observed only at voltages higher than ±150 mV; at lower voltages the channels are predominantly in a non-conducting state. Given that any potential difference across the OM will be very small if at all present, the electrophysiology data suggest that Pput2725 may not form an ion-conducting channel under physiological conditions. The relatively low conductance values suggest that even at high voltages the N-terminus may be present inside the barrel lumen, as observed in the crystal structure. By comparison, the single-channel conductance observed for NanC in the presence of 1 M KCl (~350 pS at pH 7) is almost 3-fold higher than Pput2725, and the NanC traces are also quite stable15. The ion current traces for Pput2725 are very noisy and indicate a fluctuating behavior of the narrow channel that likely reflects movement of the N-terminus within the interior of the barrel. The channel is slightly more open in terms of conductance and duration of the open state at positive voltages compared to negative voltages. At high voltages the ionic current through the channel increases linearly with applied voltage (Fig. 3B). Together the electrophysiology data indicate the presence of narrow channel in Pput2725 that requires a substantial input of energy in order to open.

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