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 potassium channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance 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, cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings reveal that voltage sensors have evolved to interact with the lipid membrane while keeping the energetic and structural perturbations to a minimum, and that water penetrates into the membrane to hydrate charged residues and shape the transmembrane electric field.

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

A combination of sequence homology analyses of mevalonate diphosphate decarboxylase (MDD) proteins and structural information for MDD leads to the hypothesis that Asp 302 and Lys 18 are active site residues in MDD. These residues were mutated to replace acidic/basic side chains and the mutant proteins were isolated and characterized. Binding and competitive displacement studies using trinitrophenyl-ATP, a fluorescent analog of substrate ATP, indicate that these mutant enzymes (D302A, D302N, K18M) retain the ability to stoichiometrically bind nucleotide triphosphates at the active site. These observations suggest the structural integrity of the mutant MDD proteins. The functional importance of mutated residues was evaluated by kinetic analysis. The 10 3 and 10 5 -fold decreases in k cat observed for the Asp 302 mutants (D302N and D302A, respectively) support assignment of a crucial catalytic role to Asp 302. A 30-fold decrease in activity and a 16-fold inflation of the K m for ATP is documented for the K18M mutant, indicating that Lys 18 influences the active site but is not crucial for reaction chemistry. Demonstration of the influence of conserved aspartate 302 appears to represent the first documentation of the functional importance of a residue in the MDD catalytic site and affords insight into phosphotransferase reactions catalyzed by a variety of enzymes in the galactokinase, homoserine kinase, mevalonate kinase, phosphomevalonate kinase (GHMP kinase) family.Keywords: mevalonate diphosphate decarboxylase; mevalonate pyrophosphate decarboxylase; GHMP kinase family; active site mapping; general base catalyst The mevalonate pathway for isoprenoid biosynthesis is utilized in selected bacteria and the reactions are also part of the sterol biosynthetic pathway in eukaryotes. Production of isopentenyl diphosphate from mevalonic acid requires three consecutive ATP-dependent reactions, the last of which is catalyzed by mevalonate diphosphate decarboxylase (MDD; EC 4.1.1.33). This enzyme catalyzes the divalent cationdependent decarboxylation of mevalonate diphosphate (also referred to as mevalonate pyrophosphate, MPP) to isopentenyl diphosphate (or isopentenyl pyrophosphate, IPP) with concurrent hydrolysis of ATP to form ADP and inorganic phosphate (Reaction 1; Bloch et al. 1959 Article published online ahead of print. Article and publication date are at