Claudio Anselmi

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.

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 Glt_Ph, 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.

Adenosine triphosphate (ATP) synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. From yeast to vertebrates, cryoelectron tomograms of these membranes have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. The rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. In this study, we present a quantitative molecular-simulation analysis that strongly suggests that the dimers of ATP synthases organize into rows spontaneously, driven by a long-range attractive force that arises from the relief of the overall elastic strain of the membrane. The strain is caused by the V-like shape of the dimers, unique among membrane protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of direct protein–protein interactions or a specific lipid composition of the membrane. We further hypothesize that, once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations by causing macroscopic membrane ridges that ultimately become the edges of cristae. In this way, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline key experiments that would be required to verify or refute this hypothesis.

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.

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.

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.

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.

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.