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We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.

Membrane transporters of the RND superfamily confer multidrug resistance to pathogenic bacteria, and are essential for cholesterol metabolism and embryonic development in humans. We use high-resolution X-ray crystallography and computational methods to delineate the mechanism of the homotrimeric RND-type proton/drug antiporter AcrB, the active component of the major efflux system AcrAB-TolC in Escherichia coli, and one most complex and intriguing membrane transporters known to date. Analysis of wildtype AcrB and four functionally-inactive variants reveals an unprecedented mechanism that involves two remote alternating-access conformational cycles within each protomer, namely one for protons in the transmembrane region and another for drugs in the periplasmic domain, 50 Å apart. Each of these cycles entails two distinct types of collective motions of two structural repeats, coupled by flanking α-helices that project from the membrane. Moreover, we rationalize how the cross-talk among protomers across the trimerization interface might lead to a more kinetically efficient efflux system. The interior of living cells is separated from their external environment by an enveloping membrane that serves as a protective barrier. To regulate the chemical composition of their interior, cells are equipped with specialized proteins in their membranes that move substances in and out of cells. Membrane proteins that expel molecules from the inside to the outside of the cell are called efflux pumps. In Escherichia coli bacteria, an efflux pump known as AcrB is part of a system that removes toxic substances from the bacterial cell—such as the antibiotics used to treat bacterial infections. AcrB and other closely related efflux pumps in pathogenic bacteria are often polyspecific transporters—they can transport a large number of different toxic molecules. These efflux pump systems are also more active in bacteria that have been targeted by antibiotics, and therefore they help bacteria to evolve resistance to multiple drugs. The emergence of bacterial multi-drug resistance is a global threat to human health; to combat this phenomenon, it is essential to understand its molecular basis. Each AcrB protein has three main parts or domains. The periplasmic domain, which is located between the two membranes that surround E. coli, works via an ‘alternating-access cycle’; that is, the shape of the periplasmic domain changes between three different forms in such a way that antibiotic molecules are first captured and subsequently squeezed through the protein towards the outside of the cell. However, the mechanism of the transmembrane domain—which is embedded in the innermost membrane of the bacterium and is the source of energy for the transport process—was not understood. Here, Eicher et al. use X-ray crystallography to examine the three-dimensional structures of the AcrB efflux pump—and several inactive variants—in high detail. Combining these results with computer simulations reveals the mechanism used by the transmembrane domain to take up protons from the exterior and transport them into the cell. Proton transport also proceeds according to an alternating-access mechanism—and, although the transmembrane and periplasmic domains are far apart, their movements are tightly linked. Thus, because proton uptake releases energy, the transmembrane domain effectively powers the periplasmic domain to expel drugs and other molecules. Eicher et al. note that a similar mechanism has not been seen before in other efflux pumps or transporter proteins. Understanding how AcrB works opens up new avenues that could be exploited to develop new drugs against multidrug resistant bacteria. Furthermore, Eicher et al. suggest that efflux pumps in humans closely related to AcrB might function in a similar way—including those required for regulation of cellular cholesterol, and for the correct development of embryos.

That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6–7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.

All rotary ATPases catalyse the interconversion of ATP and ADP-P i through a mechanism that is coupled to the transmembrane flow of H + or Na + . Physiologically, however, F/A-type enzymes specialize in ATP synthesis driven by downhill ion diffusion, while eukaryotic V-type ATPases function as ion pumps. To begin to rationalize the molecular basis for this functional differentiation, we solved the crystal structure of the Na + -driven membrane rotor of the Acetobacterium woodii ATP synthase, at 2.1 Å resolution. Unlike known structures, this rotor ring is a 9:1 heteromer of F- and V-type c-subunits and therefore features a hybrid configuration of ion-binding sites along its circumference. Molecular and kinetic simulations are used to dissect the mechanisms of Na + recognition and rotation of this c-ring, and to explain the functional implications of the V-type c-subunit. These structural and mechanistic insights indicate an evolutionary path between synthases and pumps involving adaptations in the rotor ring. Rotary ATPases exhibit similar architecture and mechanism but distinct physiological functions, operating either as ion pumps or ATP synthases. Here the authors report the structure and mechanism of the c-ring from the A. woodii ATP synthase, and suggest an evolutionary path between synthases and pumps through adaptations in this molecular rotor.

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. Humans and other mammals sense heat using a protein called the transient receptor potential vanilloid (TRPV1) channel. This protein is found in the membranes of a particular type of nerve cell, and it forms a pore that allows certain ions to pass through the membrane. Along with sensing heat, TRPV1 can also be activated by a toxin called double-knot toxin – which is found in spider venom – and by capsaicin, the active ingredient in chilli peppers. One way to investigate how a protein works is to study its three-dimensional (3D) structure. Here, Bae, Anselmi et al. use a technique called nuclear magnetic resonance (NMR) spectroscopy to produce a detailed model of the 3D structure of double-knot toxin. This model is then combined with 3D maps of TRPV1 from previous studies to predict where the toxin binds to TRPV1. This suggests that the toxin binds to a section of TRPV1 that is buried within the membrane. Moreover, the new models highlight a 'hydrophobic' region of the TRPV1 channel that may work as the heat sensor. Together, Bae, Anselmi et al.’s findings reveal a new way in which a toxin can bind to a target protein in membranes. The next step is to test the idea that the hydrophobic region identified in this work is the part of TRPV1 that senses heat.

Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs’ binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members. To date, only the open, cGMP-bound, structure of a CNG channel has been determined at atomic resolution, which is inadequate to determine the molecular events underlying gating. By using electrophysiology, site-directed mutagenesis, chemical modification, and Single Molecule Force Spectroscopy, we demonstrate that opening of CNGA1 channels is initiated by the formation of salt bridges between residues in the C-linker and S5 helix. These events trigger conformational changes of the α-helix S5, transmitted to the P-helix and leading to channel opening. Therefore, the superfamily of voltage-gated channels shares a similar molecular architecture but has evolved divergent gating mechanisms.

Bestrophins are a recently discovered family of Cl(-) channels, for which no structural information is available. Some family members are activated by increased intracellular Ca2+ concentration. Bestrophins feature a well conserved Asp-rich tract in their COOH terminus (Asp-rich domain), which is homologous to Ca2+-binding motifs in human thrombospondins and in human big-conductance Ca2+- and voltage-gated K+ channels (BK(Ca)). Consequently, the Asp-rich domain is also a candidate for Ca2+ binding in bestrophins. Based on these considerations, we constructed homology models of human bestrophin-1 (Best1) Asp-rich domain using human thrombospondin-1 X-ray structure as a template. Molecular dynamics simulations were used to identify Asp and Glu residues binding Ca2+ and to predict the effects of their mutations to alanine. We then proceeded to test selected mutations in the Asp-rich domain of the highly homologous mouse bestrophin-2. The mutants expressed in HEK-293 cells were investigated by electrophysiological experiments using the whole-cell voltage-clamp technique. Based on our molecular modeling results, we predicted that Asp-rich domain has two defined binding sites and that D301A and D304A mutations may impact the binding of the metal ions. The experiments confirmed that these mutations do actually affect the function of the protein causing a large decrease in the Ca2+-activated Cl(-) current, fully consistent with our predictions. In addition, other studied mutations (E306A, D312A) did not decrease Ca2+-activated Cl(-) current in agreement with modeling results.

Human pathogenic variants of TBC1D24 are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human TBC1D24 associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of TBC1D24 c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, Tbc1d24 expression was detected only in spiral ganglion neurons. We engineered mouse models of DFNB86 p.(Asp70Tyr) and DFNA65 p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human TBC1D24. Unexpectedly, no auditory dysfunction was detected in Tbc1d24 mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse Tbc1d24 and human TBC1D24.

Conformational fluctuations of enzymes may play an important role for substrate recognition and/or catalysis, as it has been suggested in the case of the protease enzymatic superfamily. Unfortunately, theoretically addressing this issue is a problem of formidable complexity, as the number of the involved degrees of freedom is enormous: indeed, the biological function of a protein depends, in principle, on all its atoms and on the surrounding water molecules. Here we investigated a membrane protease enzyme, the OmpT from Escherichia coli, by a hybrid molecular mechanics/coarse-grained approach, in which the active site is treated with the GROMOS force field, whereas the protein scaffold is described with a Go-model. The method has been previously tested against results obtained with all-atom simulations. Our results show that the large-scale motions and fluctuations of the electric field in the microsecond timescale may impact on the biological function and suggest that OmpT employs the same catalytic strategy as aspartic proteases. Such a conclusion cannot be drawn within the 10- to 100-ns timescale typical of current molecular dynamics simulations. In addition, our studies provide a structural explanation for the drop in the catalytic activity of two known mutants (S99A and H212A), suggesting that the coarse-grained approach is a fast and reliable tool for providing structure/function relationships for both wild-type OmpT and mutants.

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.