Dmitriy Krepkiy

Structure and hydration of membranes embedded with voltage-sensing domains

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1–S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1–S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.

Structural interactions of a voltage sensor toxin with lipid membranes.

Significance Tarantula venom contains protein toxins that interact with diverse families of ion channels and alter their activity. A number of tarantula toxins are known to interact with membranes and are thought to bind to ion channel proteins within the lipid bilayer. In the present study, we find that tarantula toxins influence the structure and dynamics of the lipid bilayer, and that the toxin orients itself within membranes to facilitate formation of the toxin–channel complexes. Our results have implications for the mechanisms of toxin action on ion channels, and more generally for protein–protein interactions within membranes. Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.

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

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. DOI: http://dx.doi.org/10.7554/eLife.11273.001

Structural Interactions between Lipids, Water and S1–S4 Voltage-Sensing Domains

Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here, we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains and that these interactions have lifetimes on the timescale of ≤ 10(-3)s. Arg residues within S1-S4 domains are well hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid headgroups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane yet are well hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage.

Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels

Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments. DOI: http://dx.doi.org/10.7554/eLife.06774.001

Expression and Purification of CB2 for NMR Studies in Micellar Solution

We demonstrate feasibility of biophysical characterization of the peripheral cannabinoid receptor CB2 produced by heterologous expression in E. coli membranes. Recombinant receptor was purified by affinity chromatography, and NMR diffusion experiments performed on CB2 solubilized in dodecylphosphocholine (DPC) micelles. Circular dichroism spectroscopy indicated high alpha-helical content (49 %) of CB2.

Engineering vanilloid-sensitivity into the rat TRPV2 channel

The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (>50°C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin(RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1. DOI: http://dx.doi.org/10.7554/eLife.16409.001

Engineering Vanilloid-Sensitivity into the TRPV2 Channel

Abstract:The TRPV1 channel is a polymodal detector of noxious stimuli, including heat, acidosis, pungent vanilloid compounds and pro-inflammatory lipids. The gating mechanisms of the closely related TRPV2 channel are poorly understood because they are vanilloid-insensitive and only activated by extremely high temperatures above 50 °C. Recent cryo-EM structures of the TRPV1 channel identified a putative vanilloid binding pocket at the interface between S1-S4 domains and the pore domain. Here we use biophysical, biochemical, and electrophysiological approaches to investigate the vanilloid binding site and gating relationships between TRPV1 and TRPV2. Although the S1-S4 domain from TRPV1 can be expressed and purified in isolation of the pore domain, it does not bind vanilloids with high affinity. We identified four non-conserved residues in the vanilloid binding pocket of TRPV1 that when introduced into TRPV2 channels are sufficient to generate high-affinity vanilloid binding and robust channel activation. Taken together, our results support the identification of the vanilloid binding pocket in the TRPV1 structure, and suggest that TRPV1 and TRPV2 channels share common gating mechanisms even though their functional properties are distinct.

Surface Characterization and Membrane Interaction of Double-Knot Toxin, an Activator of TRPV1 Channels

Double-knot toxin (DkTx) is a novel tarantula toxin that activates TRPV1 channels by binding to the extracellular pore domain of the channel, and is composed of two lobes named knot1(K1) and knot2(K2). Previous studies have shown that both lobes can be synthesized separately and activate the channel with different affinities. Recently, near atomic resolution structures of TRPV1 in distinct states (apo, capsaicin bound and DkTx&RTx bound) were reported using electron cryo-microscopy. These structures show that TRPV1 adopts a structure that is similar to Kv channels, and that the pore domain undergoes distributed conformational change upon activation in response to binding of DkTx and RTx. Although these structures show where DkTx binds, they do not have sufficient resolution to reveal the structural basis of the toxin-channel interaction. Here we solved the solution NMR structure of DkTx and dock that structure into the DkTx/RTx bound electron density maps using the Xplor-NIH program. Our results show that the toxin binds to a perimeter of the pore domain at the interface between the pore helix and S6 of neighboring subunit of the channel, and demonstrate that the toxin-channel interface is dominated by hydrophobic interactions. Interestingly, when bound to the channel, several residues on the toxin extend over the edge of the pore domain where they would be expected in interact with the surrounding membrane. To explore this possibility we tested whether DkTx can interact with membranes using a tryptophan fluorescence assay (each lobe contains a single conserved tryptophan). Indeed, each lobe of DkTx interacts with membranes, and the interaction is energetically more favorable in the bivalent toxin.

Structural Characterization of Double-Knot Toxin, an Activator of TRPV1 Channels

Venom from poisonous organisms is a rich source of peptide toxins interacting with different ion channels proteins. These peptide toxins modulate ion channels by different mechanisms, and have been widely used as tools for investigating ion channel mechanisms. Double-knot toxin (DkTx) is a novel peptide toxin that activates TRPV1 channels, and contains two inhibitory cysteine knot (ICK) motifs, as its name suggests. Previous studies show that DkTx activates TRPV1 channels, and suggest that the avidity of the toxin (slow unbinding) arises from its bivalent nature. Here we use solid-phase peptide synthesis to individually produce the two knots of DkTx (K1 and K2), fold each in vitro, and find that they exhibit different binding affinities for the channel even though they share high sequence homology. As a first step toward understanding the structural and functional relationship of DkTx binding to TRPV1 channels, we determined solution structures of each knot in using NMR. The structures show that DkTx is composed of two notably amphipathic ICK motifs (each with two beta-strands) that are connected by a flexible linker, and that K2 has a larger hydrophobic surface compared to K1. In addition, the single conserved Trp residue in each knot show different orientations, with that in K1 exhibiting greater solvent exposure. Interestingly, using intrinsic Trp fluorescence, we observe strong partitioning of DkTx and K1, but see no evidence of membrane partitioning for K2. We also made a series of K1/K2 chimeras, and identified variant residues in two loops and the C-terminus that are responsible for the higher activity of K2. From these results we propose that membrane interactions are involved in the mechanisms of DkTx activation of TRPV1, and identify surfaces of the two knots that likely involved in binding to the channel.