Explore the vast range of titles available.

Transient receptor potential (TRP) channels are multifunctional signalling molecules with many roles in sensory perception and cellular physiology. Therefore, it is not surprising that TRP channels have been implicated in numerous diseases, including hereditary disorders caused by defects in genes encoding TRP channels (TRP channelopathies). Most TRP channels are located at the cell surface, which makes them generally accessible drug targets. Early drug discovery efforts to target TRP channels focused on pain, but as our knowledge of TRP channels and their role in health and disease has grown, these efforts have expanded into new clinical indications, ranging from respiratory disorders through neurological and psychiatric diseases to diabetes and cancer. In this Review, we discuss recent findings in TRP channel structural biology that can affect both drug development and clinical indications. We also discuss the clinical promise of novel TRP channel modulators, aimed at both established and emerging targets. Last, we address the challenges that these compounds may face in clinical practice, including the need for carefully targeted approaches to minimize potential side-effects due to the multifunctional roles of TRP channels.This Review discusses recent findings in transient receptor potential (TRP) channel structural biology and the impact of these findings in drug development and clinical indications. It also addresses the challenges of modulating TRP channels and the need for targeted approaches to minimize potential side-effects due to the multifunctional roles of TRP channels.

Much structural information has been amassed on ATP-binding cassette (ABC) transporters, including hundreds of structures of isolated domains and an increasing array of full-length transporters. The structures capture different steps in the transport cycle and have aided in the design and interpretation of computational simulations and biophysics experiments. These data provide a maturing, although still incomplete, elucidation of the protein dynamics and mechanisms of substrate selection and transit through the transporters. We present an updated view of the classical alternating-access mechanism as it applies to eukaryotic ABC transporters, focusing on type I exporters. Our model helps frame the progress in, and remaining questions about, transporter energetics, how substrates are selected and how ATP is consumed to perform work at the molecular scale. Many human ABC transporters are associated with disease; we highlight progress in understanding their pharmacology through the lens of structural biology and describe how this knowledge suggests approaches to pharmacologically targeting these transporters.

Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Cells use transport proteins embedded in their membrane to acquire many of the nutrients they need to survive and grow. Different transport proteins transport different nutrients; for example, the Nramp transporters move transition metal ions across cell membranes. Nramps are found in a wide range of organisms. Bacteria use them to acquire the metals they need during the course of an infection, and humans rely on Nramps to absorb iron from food. Nramps can also transport hydrogen ions (known as protons). Understanding how the structure of an Nramp transporter changes as it transports metal ions and protons can help researchers to understand how it works. These structures can be studied using a technique called X-ray crystallography, which captures snapshots of the proteins at different stages of their task. Bozzi, Zimanyi et al. used X-ray crystallography to study the structures of an Nramp transporter from the bacterium Deinococcus radiodurans. The results reveal four of the shapes that the Nramp transporter takes on at different stages in its transport process, including the first structure to show an Nramp binding to a metal ion from the outside of the cell. Taken together, the structures suggest a new transport mechanism that has not been seen in previously studied transport proteins with similar structures. An unexpected feature of this mechanism is that Nramps transport metal ions and protons along different pathways. Studying the transport mechanisms used by Nramp transporters will help researchers to understand how cells maintain appropriate levels of metal ions, an important component of human health. The mechanisms of relatively few transport proteins are understood at a structural level, yet many share common origins and have shared characteristics. Understanding how Nramps work could therefore help us to understand how wider classes of transporters work as well.

A combination of structural, computational and biophysical tools is used to characterize the bond between tip-link proteins protocadherin 15 and cadherin 23, which have an essential role in inner-ear mechanotransduction; the bond, involving an extended protein handshake, is found to be affected by deafness mutations and is mechanically strong enough to resist forces in hair cells, adding to our understanding of hair-cell sensory transduction and interactions among cadherins. Mechanism of hair-cell sensory transduction Hair cells in the inner ear transform mechanical stimuli into electrical signals that are crucial for hearing and balance. These cells contain actin-rich stereocilia that are interconnected by filaments such as the tip link, which forms the mechanotransduction apparatus. The adhesion molecules protocadherin 15 and cadherin 23 form the tip link. In this study, David Corey et al . use a combination of structural and biophysical experiments to characterize the bond between cadherin 23 and protocadherin 15. The bond is mechanically strong enough to resist forces in hair cells and requires calcium to maintain stability. These results provide molecular insights into the mechanics of hair-cell sensory transduction. Hearing and balance use hair cells in the inner ear to transform mechanical stimuli into electrical signals 1 . Mechanical force from sound waves or head movements is conveyed to hair-cell transduction channels by tip links 2 , 3 , fine filaments formed by two atypical cadherins known as protocadherin 15 and cadherin 23 (refs 4 , 5 ). These two proteins are involved in inherited deafness 6 , 7 , 8 , 9 , 10 and feature long extracellular domains that interact tip-to-tip 5 , 11 in a Ca 2+ -dependent manner. However, the molecular architecture of this complex is unknown. Here we combine crystallography, molecular dynamics simulations and binding experiments to characterize the protocadherin 15–cadherin 23 bond. We find a unique cadherin interaction mechanism, in which the two most amino-terminal cadherin repeats (extracellular cadherin repeats 1 and 2) of each protein interact to form an overlapped, antiparallel heterodimer. Simulations predict that this tip-link bond is mechanically strong enough to resist forces in hair cells. In addition, the complex is shown to become unstable in response to Ca 2+ removal owing to increased flexure of Ca 2+ -free cadherin repeats. Finally, we use structures and biochemical measurements to study the molecular mechanisms by which deafness mutations disrupt tip-link function. Overall, our results shed light on the molecular mechanics of hair-cell sensory transduction and on new interaction mechanisms for cadherins, a large protein family implicated in tissue and organ morphogenesis 12 , 13 , neural connectivity 14 and cancer 15 .

TRPV2 is a ligand-operated temperature sensor with poorly defined pharmacology. Here, we combine calcium imaging and patch-clamp electrophysiology with cryo-electron microscopy (cryo-EM) to explore how TRPV2 activity is modulated by the phytocannabinoid Δ 9 -tetrahydrocannabiorcol (C16) and by probenecid. C16 and probenecid act in concert to stimulate TRPV2 responses including histamine release from rat and human mast cells. Each ligand causes distinct conformational changes in TRPV2 as revealed by cryo-EM. Although the binding for probenecid remains elusive, C16 associates within the vanilloid pocket. As such, the C16 binding location is distinct from that of cannabidiol, partially overlapping with the binding site of the TRPV2 inhibitor piperlongumine. Taken together, we discover a new cannabinoid binding site in TRPV2 that is under the influence of allosteric control by probenecid. This molecular insight into ligand modulation enhances our understanding of TRPV2 in normal and pathophysiology. TRPV2 is activated by temperature and cannabinoids. Here, the authors have used cryo-EM and electrophysiology to identify a cannabinoid binding site distinct from that of cannabidiol as a possible drug target for treatment of inflammation and immune-mediated diseases.

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur’s presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it—more effectively than any other amino acid—increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn 2+ , across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd 2+ -bound structure highlights differences in how Cd 2+ and Mn 2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn 2+ is different and more robust to perturbation than for transporting the toxic Cd 2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.

Most transient receptor potential (TRP) channels are regulated by phosphatidylinositol-4,5-biphosphate (PIP ₂), although the structural rearrangements occurring on PIP ₂ binding are currently far from clear. Here we report that activation of the TRP vanilloid 4 (TRPV4) channel by hypotonic and heat stimuli requires PIP ₂ binding to and rearrangement of the cytosolic tails. Neutralization of the positive charges within the sequence ¹²¹KRWRK ¹²⁵, which resembles a phosphoinositide-binding site, rendered the channel unresponsive to hypotonicity and heat but responsive to 4α-phorbol 12,13-didecanoate, an agonist that binds directly to transmembrane domains. Similar channel response was obtained by depletion of PIP ₂ from the plasma membrane with translocatable phosphatases in heterologous expression systems or by activation of phospholipase C in native ciliated epithelial cells. PIP ₂ facilitated TRPV4 activation by the osmotransducing cytosolic messenger 5′-6’-epoxyeicosatrienoic acid and allowed channel activation by heat in inside-out patches. Protease protection assays demonstrated a PIP ₂-binding site within the N-tail. The proximity of TRPV4 tails, analyzed by fluorescence resonance energy transfer, increased by depleting PIP ₂ mutations in the phosphoinositide site or by coexpression with protein kinase C and casein kinase substrate in neurons 3 (PACSIN3), a regulatory molecule that binds TRPV4 N-tails and abrogates activation by cell swelling and heat. PACSIN3 lacking the Bin-Amphiphysin-Rvs (F-BAR) domain interacted with TRPV4 without affecting channel activation or tail rearrangement. Thus, mutations weakening the TRPV4–PIP ₂ interacting site and conditions that deplete PIP ₂ or restrict access of TRPV4 to PIP ₂—in the case of PACSIN3—change tail conformation and negatively affect channel activation by hypotonicity and heat.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. To regulate the activity of proteins, cells often modify them by adding or removing chemical groups called phosphates. Therefore, the enzymes that add or remove these phosphate groups must be tightly regulated so that they are active at the right time and place. Enzymes known as phosphatases remove phosphate groups from proteins and the PP2Cs are one such family of enzymes that are found in bacteria, plants and animals. Despite their broad importance, it was not clear how cells control the PP2Cs. One way to understand how an enzyme is controlled is to compare the three-dimensional structures of the enzyme when it is active and when it is inactive. Bradshaw et al. used a PP2C enzyme from bacteria as a model to understand how the cell regulates other PP2Cs. The experiments reveal that the bacterial enzyme has a structural element that acts as a switch to control its activity. The phosphatase needs to bind metal ions to be active, and movement of the switch promotes binding of the metal ions to activate the phosphatase. The switch is also found in other members of the PP2C family. Furthermore, members of a seemingly unrelated family of enzymes called the proteasomal proteases, which degrade proteins, also have a similar architecture and are controlled by a similar switch. Thus, the phosphatase and protease families may have a common evolutionary history. Multiple members of the PP2C family are involved in cancer and other diseases. The discovery of a regulatory switch provides new opportunities to use drugs to control phosphatase activity in patients. Many cancer drugs that are currently in use or are under development target enzymes that add phosphate groups to proteins, but efforts to target the phosphatases have largely been unsuccessful. Bradshaw et al.’s findings may enable the development of new drugs that target protein phosphatases.

Protocadherins (Pcdhs) are cell adhesion and signaling proteins used by neurons to develop and maintain neuronal networks, relying on trans homophilic interactions between their extracellular cadherin (EC) repeat domains. We present the structure of the antiparallel EC1-4 homodimer of human PcdhγB3, a member of the γ subfamily of clustered Pcdhs. Structure and sequence comparisons of α, β, and γ clustered Pcdh isoforms illustrate that subfamilies encode specificity in distinct ways through diversification of loop region structure and composition in EC2 and EC3, which contains isoform-specific conservation of primarily polar residues. In contrast, the EC1/EC4 interface comprises hydrophobic interactions that provide non-selective dimerization affinity. Using sequence coevolution analysis, we found evidence for a similar antiparallel EC1-4 interaction in non-clustered Pcdh families. We thus deduce that the EC1-4 antiparallel homodimer is a general interaction strategy that evolved before the divergence of these distinct protocadherin families. As the brain develops, nerve cells or neurons connect with one another to form complex networks. These connections form between branch-like structures, called dendrites, that project from the cell body of each neuron. To prevent unneeded connections from forming, dendrites that belong to the same neuron need a way to recognize and avoid one another. A family of proteins called protocadherins supports this process of self-avoidance. Protocadherins have three main parts or domains: an extracellular domain that faces outwards away from the cell, a transmembrane domain that sits within the cell’s surface membrane and an intracellular domain that faces into the cell’s interior. There are two major groups of protocadherins – clustered and non-clustered – and the former are responsible for the self-avoidance behavior between dendrites. Clustered protocadherins in turn comprise three subfamilies, each of which consists of multiple variants with slightly different structures (known as isoforms). The particular set of protocadherin isoforms that a neuron displays on its surface distinguishes that neuron from all others, a little like a barcode. When two dendrites meet, the protocadherins in their membranes come into contact with one another. If both dendrites come from the same neuron and therefore possess identical sets of protocadherins, then all protocadherins can form two-subunit complexes containing one copy of the same isoform from each dendrite. These complexes are called homodimers and their formation acts as a signal that informs the cell that it has encountered one of its own dendrites and should therefore not establish a connection. By using X-rays to determine the structure of a crystallized protocadherin fragment down to the level of its individual atoms, Nicoludis et al. now reveal exactly how clustered protocadherins form homodimers. The results show that each protocadherin subfamily uses a slightly different type of interaction due to differences in the structure of their extracellular domains. The next challenge is to identify the signaling cascade that is triggered by the formation of clustered protocadherin homodimers, and to work out how activation of this cascade prevents a permanent connection from forming. In addition, the results of Nicoludis et al. predict that some non-clustered protocadherins form dimers with a similar architecture to that of clustered protocadherins. This possibility should also be tested experimentally.