Protein-Lipid Interfaces Can Drive the Functions of Membrane-Embedded Protein-Protein Complexes

Abstract

The roles of surrounding membrane lipids in the functions of transmembrane and peripheral membrane proteins are largely unknown. Herein, we utilize the recently reported structures of the TRPV1 ion channel protein bound to its potent protein agonist, the double-knot toxin (DkTx), as a model system to investigate the roles of toxin-lipid interfaces in TRPV1 activation by characterizing a series of DkTx variants electrophysiologically. Together with membrane partitioning experiments, these studies reveal that toxin-lipid interfaces play an overwhelmingly dominant role in channel activation as compared to lipid-devoid toxin-channel interfaces. Additionally, we find that whereas the membrane interfaces formed by one of the knots of the toxin endow it with its low channel-dissociation rate, those formed by other knot contribute primarily to its potency. These studies establish that protein-lipid interfaces play nuanced yet profound roles in the function of protein-protein complexes within membranes.

Figure 1

DkTx–TRPV1 complex. (a) The DkTx–lipid–TRPV1 tripartite complex viewed sideways from within the membrane, PDB: 5irx, and a zoomed-in view of the complex depicting the pore domains of three channel monomers (right panel; the fourth monomer is not depicted to enhance clarity). The solid horizontal lines represent the membrane boundaries (the region above the top line is extracellular, and the region below the bottom line is intracellular). (b) Structure of DkTx depicting residues that interact exclusively with TRPV1 residues and not with lipids in green and those that interact with lipids in blue. Only the lipid-interacting residues are labeled. The same color code is used to denote the sequence of the K1 and K2 knots of DkTx at the bottom. The residues of loops 2 and 4 of each knot are boxed. (c) A two-electrode voltage clamp electrophysiology recording of TRPV1 depicting activation by capsaicin (Caps, 5 μM) and subsequently by DkTx (3.3 μM), followed by wash-off for 30 min, and ultimately complete channel block by ruthenium red (RR, 7 μM). The dotted line represents zero current.

Figure 2

Effects on the potency of DkTx for TRPV1 activation upon alteration of protein–protein and protein–lipid interfaces. (a) Dose–response plots of the variants of exclusively channel-interacting (top) and lipid-interacting (bottom) residues of DkTx. The K1 knot variants of DkTx are depicted on the left and K2 variants on the right. (b) EC₅₀ values of all DkTx variants. The bars in green correspond to variants of exclusively channel-interacting DkTx residues, and the ones in blue are of those that interact with lipids. The EC₅₀ values for the W53A and W53L variants could not be obtained owing to their extremely low potency (this is denoted by the arrows on the top of the bars corresponding to these variants). “WT” is an abbreviation for “wild-type”. Each data point is an average of three to five recordings, and the error bars correspond to standard deviation values.

Figure 3

Toxin wash-off studies. (a) Electrophysiological recordings obtained for fast washing-off DkTx variants. Capsaicin was applied at a concentration of 5 μM, and the toxins were applied at 6.6 μM, except for V8A, which was tested at 13.2 μM. (b) Wash-off studies on wild-type DkTx showing fast wash-off when applied at a low concentration (top panel) and concentration dependence of wash-off kinetics (middle and bottom panels). The y axis of the plot on the bottom panel depicts % reduction in current after 3 min of buffer perfusion post wild-type DkTx-mediated channel activation. The blue line shown was generated by fitting a linear equation to the data. (c) Averaged wash-off current traces obtained for wild-type DkTx and fast washing-off lipid-interacting K1 variants of DkTx (left panel) and those of their corresponding K2 variants (right panel). (d) Bar graph depicting the percentage reduction in current after 3 min of buffer perfusion post-toxin-mediated channel activation for all variants (bars corresponding to the variants of lipid-independent channel-interacting residues are depicted in green and those for lipid-interacting residues are depicted in blue). The experiments (in both c and d) were performed at saturation concentrations of the respective toxins denoted within parentheses in Figure 3d. Each current trace/data point is an average of three to five recordings, and the error bars correspond to standard deviation values.

Figure 4

Toxin–membrane interaction studies on DkTx and its variants by employing tryptophan fluorescence (a–c) and oocyte depletion (d,e). (a) Tryptophan emission spectra of wild-type DkTx (black) and the W11A variant (red) in the presence of 1:1 POPC–POPG liposomes (total lipid concentration: 0.1 mM) have been depicted as solid curves, whereas the ones obtained in the absence of lipids are shown as dashed curves. (b) Plots of normalized relative fluorescence intensity (F/F_o) at 320 nm for the wild-type toxin and the variants of analogous phenylalanine and tryptophan residues of the K1 and K2 knots as a function of the available lipid concentration. (c) Plot of % wash-off of wild-type DkTx and K1 variants (solid squares), and those of K2 variants (open squares) after 3 min of buffer perfusion post TRPV1 activation by saturation concentrations of the toxins (obtained from the electrophysiology experiments) versus mol. fraction partitioning coefficients (K_x) values (obtained from the tryptophan fluorescence experiments). The blue line shown was generated by fitting a linear equation to the data for the K1 variants. (d) HPLC traces depicting toxin depletion upon incubation of DkTx and its variants with Xenopus laevis oocytes. Traces in black correspond to the controls wherein the toxins solubilized in buffer devoid of oocytes were subjected to HPLC analysis, whereas those in red were obtained when the supernatants of toxin solutions incubated with 100 oocytes were subjected to HPLC. (e) Plot of % wash-off of wild-type DkTx and K1 variants (solid squares) and K2 variants (open squares) after 3 min of buffer perfusion post TRPV1 activation by saturation concentrations of the toxins (obtained from the electrophysiology experiments) versus fractional depletion (obtained from HPLC peak areas as described in the Supporting Information section). The blue line shown was generated by fitting a linear equation to the data for the K1 variants. Each data point is an average of three to five recordings/assays, and the error bars correspond to standard deviation values.

Figure 5

Plots of potency for TRPV1 activation (EC₅₀ values) versus channel-dissociation rates (% wash-off), of DkTx variants. (a) DkTx variants of the K1 knot residues and (b) those of the K2 knot residues. Data points for the variants of lipid-interacting residues are depicted in blue, those of channel-interacting residues in green, and that for the wild-type toxin in black. Percentage wash-off depicts the % reduction in current after 3 min of buffer perfusion post channel activation with saturation concentrations of the toxin variants. The dotted lines represent the data for wild-type DkTx.

Figure 6

Functionally critical toxin residues of the (a) K1 knot and (b) K2 knot, identified in this study mapped onto the structure (pdb: 5irx(1)) along with the TRPV1 residues and lipid molecules they interact with. The left panels depict the toxin residues in blue, the lipids in the sticks representation, and the channel residues in the color employed to depict the channel subunit they belong to (the color scheme used to render the channel subunits is the same one that was used in Figure 1). The right panels depict the same interface shown on the left panel in the surface orientation rendered by employing the Eisenberg scale, wherein the intensity of the red color is proportional to the hydrophobicity.

Figure 7

Our proposed “toxin relay” model that explains the slow wash-off feature of DkTx. DkTx is represented by a dumbbell-shaped object with its two ellipses denoting the two knots of the toxin. The knots of the toxin molecules that do not bind to the channel are depicted as empty ellipses and those that bind to the channel as filled ellipses. The wavy blue curves depict buffer flow.