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Gating topology of the proton-coupled oligopeptide symporters.

Fowler PW, Orwick-Rydmark M, Radestock S, Solcan N, Dijkman PM, Lyons JA, Kwok J, Caffrey M, Watts A, Forrest LR, Newstead S - Structure (2015)

Bottom Line: Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters.Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood.Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: philip.fowler@bioch.ox.ac.uk.

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The Kink Produced by the Conserved Prolines in H8 Is Important for Transport(A) In the inward-occluded experimental structure of PepTSo, H8 (in red) is kinked because of two prolines, P345 and P353 (in pink). We measured the relative motion of the C-terminal ends of H8 and H6 (pink) by attaching MTSL spin labels to the E364C R201C mutant of PepTSo.(B) The same features are highlighted in blue on the outward-open model of PepTSo, demonstrating that this model predicts a shorter distance between positions 201 and 364.(C) There is moderate overlap between the R201C E364C spin-spin distance distributions measured experimentally (black line) and those predicted from the inward-occluded crystal structure of PepTSo (filled red bars). The outward-open model instead predicts a shorter distance between the ends of H6 and H8 (filled blue bars).(D) Mutating both prolines to alanine results in a more complex spin-spin distance distribution. We suggest that H8 in the R201C E364C P345A P353A is straighter than wild-type. Consistent with this, there is now reasonable agreement between the spin-spin distance distributions measured experimentally (black line) and those predicted from the model of the outward-facing conformation (filled blue bars).(E) Mutating either or both prolines in PepTSo or PepTSt either reduces or abolishes proton-driven active transport.Error bars indicate the standard deviations from triplicate experiments.
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fig8: The Kink Produced by the Conserved Prolines in H8 Is Important for Transport(A) In the inward-occluded experimental structure of PepTSo, H8 (in red) is kinked because of two prolines, P345 and P353 (in pink). We measured the relative motion of the C-terminal ends of H8 and H6 (pink) by attaching MTSL spin labels to the E364C R201C mutant of PepTSo.(B) The same features are highlighted in blue on the outward-open model of PepTSo, demonstrating that this model predicts a shorter distance between positions 201 and 364.(C) There is moderate overlap between the R201C E364C spin-spin distance distributions measured experimentally (black line) and those predicted from the inward-occluded crystal structure of PepTSo (filled red bars). The outward-open model instead predicts a shorter distance between the ends of H6 and H8 (filled blue bars).(D) Mutating both prolines to alanine results in a more complex spin-spin distance distribution. We suggest that H8 in the R201C E364C P345A P353A is straighter than wild-type. Consistent with this, there is now reasonable agreement between the spin-spin distance distributions measured experimentally (black line) and those predicted from the model of the outward-facing conformation (filled blue bars).(E) Mutating either or both prolines in PepTSo or PepTSt either reduces or abolishes proton-driven active transport.Error bars indicate the standard deviations from triplicate experiments.

Mentions: We previously noted the poor agreement between the DEER E201C-R364C distance distributions and our predictions from the outward-facing PepTSo model (Figure S3). This pair of residues reports the relative motions of H6 and H8, respectively (Figure 8A), which are part of repeat units B and C, respectively. A major structural difference between repeat units C and D is the kink in H8, which is absent in its symmetry-related partner, H11. Examining the structure of PepTSo suggests that the kink in H8 is due to two prolines, P345 and P353 (P329 and P345 in PepTSt), the latter being highly conserved across the POT family (Figure S8). Prolines are known to favor kinks in transmembrane helices (Fowler and Sansom, 2013). Mutating both prolines to alanine led to an altered spin-spin distance distribution in PepTSo with two peaks, one at a position similar to that of the single peak observed in wild-type, and another, more dominant, peak at a shorter distance (Figure 8D; Figure S3). This second peak overlaps with the distance distribution predicted from the outward-facing model and is likely due to a straightening of H8 caused by the removal of the two proline residues, resulting in a decrease in the distance between the intracellular ends of H6 and H8. We propose that the anomalously short distance predicted by the outward-open model of PepTSo is the result of the repeat-swapping process making H8 too straight. To support this hypothesis, mutating the first proline in either PepTSo (P345A) or PepTSt (P329A) reduced proton-driven transport (Figure 8E). The effect was more pronounced when the second, more conserved proline was mutated; the P353A PepTSo mutant abolished active transport and the P345A PepTSt mutant had only 20% of the level of transport activity of the wild-type. The PepTSo double mutant had no detectable transport activity, whereas the PepTSt double mutant had activity similar to that of the P329A mutant.


Gating topology of the proton-coupled oligopeptide symporters.

Fowler PW, Orwick-Rydmark M, Radestock S, Solcan N, Dijkman PM, Lyons JA, Kwok J, Caffrey M, Watts A, Forrest LR, Newstead S - Structure (2015)

The Kink Produced by the Conserved Prolines in H8 Is Important for Transport(A) In the inward-occluded experimental structure of PepTSo, H8 (in red) is kinked because of two prolines, P345 and P353 (in pink). We measured the relative motion of the C-terminal ends of H8 and H6 (pink) by attaching MTSL spin labels to the E364C R201C mutant of PepTSo.(B) The same features are highlighted in blue on the outward-open model of PepTSo, demonstrating that this model predicts a shorter distance between positions 201 and 364.(C) There is moderate overlap between the R201C E364C spin-spin distance distributions measured experimentally (black line) and those predicted from the inward-occluded crystal structure of PepTSo (filled red bars). The outward-open model instead predicts a shorter distance between the ends of H6 and H8 (filled blue bars).(D) Mutating both prolines to alanine results in a more complex spin-spin distance distribution. We suggest that H8 in the R201C E364C P345A P353A is straighter than wild-type. Consistent with this, there is now reasonable agreement between the spin-spin distance distributions measured experimentally (black line) and those predicted from the model of the outward-facing conformation (filled blue bars).(E) Mutating either or both prolines in PepTSo or PepTSt either reduces or abolishes proton-driven active transport.Error bars indicate the standard deviations from triplicate experiments.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321885&req=5

fig8: The Kink Produced by the Conserved Prolines in H8 Is Important for Transport(A) In the inward-occluded experimental structure of PepTSo, H8 (in red) is kinked because of two prolines, P345 and P353 (in pink). We measured the relative motion of the C-terminal ends of H8 and H6 (pink) by attaching MTSL spin labels to the E364C R201C mutant of PepTSo.(B) The same features are highlighted in blue on the outward-open model of PepTSo, demonstrating that this model predicts a shorter distance between positions 201 and 364.(C) There is moderate overlap between the R201C E364C spin-spin distance distributions measured experimentally (black line) and those predicted from the inward-occluded crystal structure of PepTSo (filled red bars). The outward-open model instead predicts a shorter distance between the ends of H6 and H8 (filled blue bars).(D) Mutating both prolines to alanine results in a more complex spin-spin distance distribution. We suggest that H8 in the R201C E364C P345A P353A is straighter than wild-type. Consistent with this, there is now reasonable agreement between the spin-spin distance distributions measured experimentally (black line) and those predicted from the model of the outward-facing conformation (filled blue bars).(E) Mutating either or both prolines in PepTSo or PepTSt either reduces or abolishes proton-driven active transport.Error bars indicate the standard deviations from triplicate experiments.
Mentions: We previously noted the poor agreement between the DEER E201C-R364C distance distributions and our predictions from the outward-facing PepTSo model (Figure S3). This pair of residues reports the relative motions of H6 and H8, respectively (Figure 8A), which are part of repeat units B and C, respectively. A major structural difference between repeat units C and D is the kink in H8, which is absent in its symmetry-related partner, H11. Examining the structure of PepTSo suggests that the kink in H8 is due to two prolines, P345 and P353 (P329 and P345 in PepTSt), the latter being highly conserved across the POT family (Figure S8). Prolines are known to favor kinks in transmembrane helices (Fowler and Sansom, 2013). Mutating both prolines to alanine led to an altered spin-spin distance distribution in PepTSo with two peaks, one at a position similar to that of the single peak observed in wild-type, and another, more dominant, peak at a shorter distance (Figure 8D; Figure S3). This second peak overlaps with the distance distribution predicted from the outward-facing model and is likely due to a straightening of H8 caused by the removal of the two proline residues, resulting in a decrease in the distance between the intracellular ends of H6 and H8. We propose that the anomalously short distance predicted by the outward-open model of PepTSo is the result of the repeat-swapping process making H8 too straight. To support this hypothesis, mutating the first proline in either PepTSo (P345A) or PepTSt (P329A) reduced proton-driven transport (Figure 8E). The effect was more pronounced when the second, more conserved proline was mutated; the P353A PepTSo mutant abolished active transport and the P345A PepTSt mutant had only 20% of the level of transport activity of the wild-type. The PepTSo double mutant had no detectable transport activity, whereas the PepTSt double mutant had activity similar to that of the P329A mutant.

Bottom Line: Proton-coupled oligopeptide transporters belong to the major facilitator superfamily (MFS) of membrane transporters.Recent crystal structures suggest the MFS fold facilitates transport through rearrangement of their two six-helix bundles around a central ligand binding site; how this is achieved, however, is poorly understood.Using modeling, molecular dynamics, crystallography, functional assays, and site-directed spin labeling combined with double electron-electron resonance (DEER) spectroscopy, we present a detailed study of the transport dynamics of two bacterial oligopeptide transporters, PepTSo and PepTSt. Our results identify several salt bridges that stabilize outward-facing conformations and we show that, for all the current structures of MFS transporters, the first two helices of each of the four inverted-topology repeat units form half of either the periplasmic or cytoplasmic gate and that these function cooperatively in a scissor-like motion to control access to the peptide binding site during transport.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: philip.fowler@bioch.ox.ac.uk.

Show MeSH