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Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin.

Bae C, Anselmi C, Kalia J, Jara-Oseguera A, Schwieters CD, Krepkiy D, Won Lee C, Kim EH, Kim JI, Faraldo-Gómez JD, Swartz KJ - Elife (2016)

Bottom Line: We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes.Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation.Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

No MeSH data available.


Computational alanine-scan of the DkTx-TRPV1 interface.The plot reports the estimated change in the association free energy of the toxin-channel complex upon alanine substitution of each of the residues in the toxin, calculated with ROSETTA (see Methods). Positive values indicate a destabilizing effect. The estimates are averages over 200,000 snapshots of the complex extracted from a MD simulation trajectory in which the experimental cryo-EM map is used as a three-dimensional restraint.DOI:http://dx.doi.org/10.7554/eLife.11273.018
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fig4s2: Computational alanine-scan of the DkTx-TRPV1 interface.The plot reports the estimated change in the association free energy of the toxin-channel complex upon alanine substitution of each of the residues in the toxin, calculated with ROSETTA (see Methods). Positive values indicate a destabilizing effect. The estimates are averages over 200,000 snapshots of the complex extracted from a MD simulation trajectory in which the experimental cryo-EM map is used as a three-dimensional restraint.DOI:http://dx.doi.org/10.7554/eLife.11273.018

Mentions: To investigate the interaction between the toxin and channel in more detail, we carried out an all-atom MD simulation of the complex embedded in a phospholipid bilayer (Figure 4A). To enhance the exploration of diverse interaction patterns in a limited simulation time (~500 ns), we coupled the χ1 and χ2 torsion angles of all interfacial side-chains in the toxin and channel to a fictitious high-temperature bath, using an extended-Lagrangian approach (Iannuzzi et al., 2003) (see Methods). To preclude the dissociation of the complex under this bias, the cryo-EM envelop was used as a three-dimensional restraint. A contact map between residues in DkTx and the outer pore of TRPV1 was then generated from the last 200 ns of simulation, so as to identify the most pronounced interactions (Figure 4D). Two regions of the extracellular surface of the channel stand out as forming the most persistent side-chain contacts with either of the two lobes of DkTx, namely the N-terminus of the pore-helix, primarily via Y631, and a stretch of the pore-loop and N-terminus of S6, including N652, D654, F655, K656, A657 and V658; additional interactions are mediated by K535 and E536, in S4 (Figure 4D; Figure 4—figure supplement 1; Video 2). The contacts with the pore and S6 helices are particularly worth noting because the A657P mutation effectively abolishes channel activation by DkTx, while Y631A enhances it (Bohlen et al., 2010). Many of the contacts on the toxin are with residues that are equivalent in K1 and K2, e.g. W11 and W53, F27 and F67, K14 and K56 and G12 and G54, respectively, and a computational alanine-scanning (Ala-scan) analysis of the toxin-channel interface, based on the configurations explored during the MD simulation (see Methods), indicate that these are all influential (Figure 4—figure supplement 2). However, there are also differences between K1 and K2, which might underlie their different affinity for TRPV1. The most interesting unique interactions for K2 involve S55 (K13 in K1), L65 (M25 in K1) and R75 (K35 in K1). The persistence of these interactions is worth noting because these residues are located in either loop 2 (S55), loop 4 (L65) or the C-terminus (R75), and transfer of these segments from K2 into K1 yields a pronounced increase in the binding affinity of isolated K1 (Figure 3F). These residues are also predicted to have a significant stabilizing effect by the computational Ala-scan (Figure 4—figure supplement 2).10.7554/eLife.11273.016Figure 4.DkTx interactions with TRPV1 and the surrounding lipid bilayer from MD simulations.


Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin.

Bae C, Anselmi C, Kalia J, Jara-Oseguera A, Schwieters CD, Krepkiy D, Won Lee C, Kim EH, Kim JI, Faraldo-Gómez JD, Swartz KJ - Elife (2016)

Computational alanine-scan of the DkTx-TRPV1 interface.The plot reports the estimated change in the association free energy of the toxin-channel complex upon alanine substitution of each of the residues in the toxin, calculated with ROSETTA (see Methods). Positive values indicate a destabilizing effect. The estimates are averages over 200,000 snapshots of the complex extracted from a MD simulation trajectory in which the experimental cryo-EM map is used as a three-dimensional restraint.DOI:http://dx.doi.org/10.7554/eLife.11273.018
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764579&req=5

fig4s2: Computational alanine-scan of the DkTx-TRPV1 interface.The plot reports the estimated change in the association free energy of the toxin-channel complex upon alanine substitution of each of the residues in the toxin, calculated with ROSETTA (see Methods). Positive values indicate a destabilizing effect. The estimates are averages over 200,000 snapshots of the complex extracted from a MD simulation trajectory in which the experimental cryo-EM map is used as a three-dimensional restraint.DOI:http://dx.doi.org/10.7554/eLife.11273.018
Mentions: To investigate the interaction between the toxin and channel in more detail, we carried out an all-atom MD simulation of the complex embedded in a phospholipid bilayer (Figure 4A). To enhance the exploration of diverse interaction patterns in a limited simulation time (~500 ns), we coupled the χ1 and χ2 torsion angles of all interfacial side-chains in the toxin and channel to a fictitious high-temperature bath, using an extended-Lagrangian approach (Iannuzzi et al., 2003) (see Methods). To preclude the dissociation of the complex under this bias, the cryo-EM envelop was used as a three-dimensional restraint. A contact map between residues in DkTx and the outer pore of TRPV1 was then generated from the last 200 ns of simulation, so as to identify the most pronounced interactions (Figure 4D). Two regions of the extracellular surface of the channel stand out as forming the most persistent side-chain contacts with either of the two lobes of DkTx, namely the N-terminus of the pore-helix, primarily via Y631, and a stretch of the pore-loop and N-terminus of S6, including N652, D654, F655, K656, A657 and V658; additional interactions are mediated by K535 and E536, in S4 (Figure 4D; Figure 4—figure supplement 1; Video 2). The contacts with the pore and S6 helices are particularly worth noting because the A657P mutation effectively abolishes channel activation by DkTx, while Y631A enhances it (Bohlen et al., 2010). Many of the contacts on the toxin are with residues that are equivalent in K1 and K2, e.g. W11 and W53, F27 and F67, K14 and K56 and G12 and G54, respectively, and a computational alanine-scanning (Ala-scan) analysis of the toxin-channel interface, based on the configurations explored during the MD simulation (see Methods), indicate that these are all influential (Figure 4—figure supplement 2). However, there are also differences between K1 and K2, which might underlie their different affinity for TRPV1. The most interesting unique interactions for K2 involve S55 (K13 in K1), L65 (M25 in K1) and R75 (K35 in K1). The persistence of these interactions is worth noting because these residues are located in either loop 2 (S55), loop 4 (L65) or the C-terminus (R75), and transfer of these segments from K2 into K1 yields a pronounced increase in the binding affinity of isolated K1 (Figure 3F). These residues are also predicted to have a significant stabilizing effect by the computational Ala-scan (Figure 4—figure supplement 2).10.7554/eLife.11273.016Figure 4.DkTx interactions with TRPV1 and the surrounding lipid bilayer from MD simulations.

Bottom Line: We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes.Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation.Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

No MeSH data available.