Mouse Models of Human Pathogenic Variants of TBC1D24 Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness.

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.

Mitochondrial Enzymes of the Urea Cycle Cluster at the Inner Mitochondrial Membrane.

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein-"variable segment," which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.

Large-scale state-dependent membrane remodeling by a transporter protein.

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.

Mitochondrial ATP synthase dimers spontaneously associate due to a long-range membrane-induced force

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.

The gating mechanism in cyclic nucleotide-gated ion channels.

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.

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.

High-resolution structure and mechanism of an F/V-hybrid rotor ring in a Na⁺-coupled ATP synthase.

All rotary ATPases catalyse the interconversion of ATP and ADP-Pi 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.

Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB.

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.

Dynamics of the antigen-binding grooves in CD1 proteins: reversible hydrophobic collapse in the lipid-free state.

CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a meta-analysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two α-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these α-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal α-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization.

Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.

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.

Insights on the acetylated NF-κB transcription factor complex with DNA from molecular dynamics simulations.

The nuclear factor-κB (NF-κB) is a DNA sequence-specific regulator of many important biological processes, whose activity is modulated by enzymatic acetylation. In one of the best functionally characterized NF-κB complexes, the p50/p65 heterodimer, acetylation of K221 at p65 causes a decrease of DNA dissociation rate, whilst the acetylation of K122 and K123, also at p65, markedly decreases the binding affinity for DNA. By means of molecular dynamics simulations based on the X-ray structure of the p50/p65 complex with DNA, we provide insights on the structural determinants of the acetylated complexes in aqueous solution. Lysine acetylation involves the loss of favorable electrostatic interactions between DNA and NF-κB, which is partially compensated by the reduction of the desolvation free-energy of the two binding partners. Acetylation at both positions K122 and K123 is associated with a decrease of the electrostatic potential at the p65/DNA interface, which is only partially counterbalanced by an increase of the local Na(+) concentration. It induces the disruption of base-specific and nonspecific interactions between DNA and NF-κB and it is consistent with the observed decrease of binding affinity. In contrast, acetylation at position K221 results in the loss of nonspecific protein-DNA interactions, but the DNA recognition sites are not affected. In addition, the loss of protein-DNA interactions is likely to be counterbalanced by an increase of the configurational entropy of the complex, which provides, at a speculative level, a justification for the observed decrease of NF-κB/DNA dissociation rate.

GRIFFIN: A versatile methodology for optimization of protein-lipid interfaces for membrane protein simulations.

As new atomic structures of membrane proteins are resolved, they reveal increasingly complex transmembrane topologies, and highly irregular surfaces with crevices and pores. In many cases, specific interactions formed with the lipid membrane are functionally crucial, as is the overall lipid composition. Compounded with increasing protein size, these characteristics pose a challenge for the construction of simulation models of membrane proteins in lipid environments; clearly, that these models are sufficiently realistic bears upon the reliability of simulation-based studies of these systems. Here, we introduce GRIFFIN, which uses a versatile framework to automate and improve a widely-used membrane-embedding protocol. Initially, GRIFFIN carves out lipid and water molecules from a volume equivalent to that of the protein, so as to conserve the system density. In the subsequent optimization phase GRIFFIN adds an implicit grid-based protein force-field to a molecular dynamics simulation of the pre-carved membrane. In this force-field, atoms inside the implicit protein volume experience an outward force that will expel them from that volume, whereas those outside are subject to electrostatic and van-der-Waals interactions with the implicit protein. At each step of the simulation, these forces are updated by GRIFFIN and combined with the intermolecular forces of the explicit lipid-water system. This procedure enables the construction of realistic and reproducible starting configurations of the protein-membrane interface within a reasonable timeframe and with minimal intervention. GRIFFIN is a standalone tool designed to work alongside any existing molecular dynamics package, such as NAMD or GROMACS.

Mechanism of substrate shuttling by the acyl-carrier protein within the fatty acid mega-synthase.

Fatty acid mega-synthases (FAS) are large complexes that integrate into a common protein scaffold all the enzymes required for the elongation of aliphatic chains. In fungi, FAS features two independent dome-shaped structures, each 3-fold symmetric, that serve as reaction chambers. Inside each chamber, three acyl-carrier proteins (ACP) are found double-tethered to the FAS scaffold by unstructured linkers; these are believed to shuttle the substrate among catalytic sites by a mechanism that is yet unknown. We present a computer-simulation study of the mechanism of ACP substrate-shuttling within the FAS reaction chamber, and a systematic assessment of the influence of several structural and energetic factors thereon. Contrary to earlier proposals, the ACP dynamics appear not to be hindered by the length or elasticity of the native linkers, nor to be confined in well-defined trajectories. Instead, each ACP domain may reach all catalytic sites within the reaction chamber, in a manner that is essentially stochastic. Nevertheless, the mechanism of ACP shuttling is clearly modulated by volume-exclusion effects due to molecular crowding and by electrostatic steering toward the chamber walls. Indeed, the probability of ACP encounters with equivalent catalytic sites was found to be asymmetric. We show how this intriguing asymmetry is an entropic phenomenon that arises from the steric hindrance posed by the ACP linkers when extended across the chamber. Altogether, these features provide a physically realistic rationale for the emergence of substrate-shuttling compartmentalization and for the apparent functional advantage of the spatial distribution of the catalytic centers.

Insight into the structure of an endopolygalacturonase from the phytopathogen Burkholderia cepacia: a biochemical and computational study.

We have recently investigated and characterized the mode of action of BcPeh28A, an endopolygalacturonase (endoPG) from the phytopathogen Burkholderia cepacia. EndoPGs belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. Here we report a 3-D structural model of BcPeh28A by combining mass spectrometry (MS) analysis, aimed at disulphide bridges mapping, and computational modelling tools. MS analyses have revealed the complete pattern of disulphide bridges in BcPeh28A, pointing out the presence of three disulphide bonds, defined as Cys3-25, Cys216-244 and Cys309-421. A 3-D model of BcPeh28A was generated by computational methods based on profile-profile sequence alignments and fold recognition algorithms. The final model exhibits a right-handed ß-helix fold with eleven ß-helical coils and includes the disulphide bonds as additional spatial restraints. Molecular dynamics simulations have been performed to test the conformational stability of the model. Finally, the structural analysis of the BcPeh28A model allows defining the architecture and the amino acid topology of the subsites involved in the catalysis and in the substrate binding specificity.

Regulation of bestrophins by Ca2+: a theoretical and experimental study.

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.

The analysis of desensitizing CNGA1 channels reveals molecular interactions essential for normal gating.

The pore region of cyclic nucleotide-gated (CNG) channels acts as the channel gate. Therefore, events occurring in the cyclic nucleotide-binding (CNB) domain must be coupled to the movements of the pore walls. When Glu363 in the pore region, Leu356 and Thr355 in the P helix, and Phe380 in the upper portion of the S6 helix are mutated into an alanine, gating is impaired: mutant channels E363A, L356A, T355A, and F380A desensitize in the presence of a constant cGMP concentration, contrary to what can be observed in wild-type (WT) CNGA1 channels. Similarly to C-type inactivation of K(+) channels, desensitization in these mutant channels is associated with rearrangements of residues in the outer vestibule. In the desensitized state, Thr364 residues in different subunits become closer and Pro366 becomes more accessible to extracellular reagents. Desensitization is also observed in the mutant channel L356C, but not in the double-mutant channel L356C+F380C. Mutant channels L356F and F380K did not express, but cGMP-gated currents with a normal gating were observed in the double-mutant channels L356F+F380L and L356D+F380K. Experiments with tandem constructs with L356C, F380C, and L356C+F380C and WT channels indicate that the interaction between Leu356 and Phe380 is within the same subunit. These results show that Leu356 forms a hydrophobic interaction with Phe380, coupling the P helix with S6, whereas Glu363 could interact with Thr355, coupling the pore wall to the P helix. These interactions are essential for normal gating and underlie the transduction between the CNB domain and the pore.

Movements of native C505 during channel gating in CNGA1 channels.

We investigated conformational changes occurring in the C-linker and cyclic nucleotide-binding (CNB) domain of CNGA1 channels by analyzing the inhibition induced by thiol-specific reagents in mutant channels Q409C and A414C in the open and closed state. Cd(2+) (200 microM) inhibited irreversibly mutant channels Q409C and A414C in the closed but not in the open state. Cd(2+) inhibition was abolished in the mutant A414C(cys-free), in the double mutant A414C + C505T and in the tandem construct A414C + C505T/CNGA1, but it was present in the construct A414C + C505(cys-free). The cross-linker reagent M-2-M inhibited mutant channel Q409C in the open state. M-2-M inhibition in the open state was abolished in the double mutant Q409C + C505T and in the tandem construct Q409C + C505T/CNGA1. These results show that C(alpha) of C505 in the closed state is located at a distance between 4 and 10.5 A from the C(alpha) of A414 of the same subunit, but in the open state C505 moves towards Q409 of the same subunit at a distance that ranges from 10.5 to 12.3 A from C(alpha) of this residue. These results are not consistent with a 3-D structure of the CNGA1 channel homologous to the structure of HCN2 channels either in the open or in the closed state.

Molecular dissection of human oncostatin M-mediated signal transductions through site-directed mutagenesis.

The binding of oncostatin M (OM) to type I and type II receptor complexes elicits various biological responses by activating MEK/ERK and JAK/STAT signaling pathways. Some OM effects are clinically desirable such as reducing hyperlipidemia through the activation of hepatic LDL receptor transcription, a downstream event of ERK activation. The OM pro-inflammatory responses via induction of acute phase protein gene expression have been associated with STAT activation. In this study, by conducting site-directed mutagenesis, bioassays and molecular modeling we have defined 4 OM residues that are differently involved in the activation of ERK or STAT signaling pathway in HepG2 cells. We show that mutation of Lys163 to alanine totally abolished OM-mediated signaling, possibly because such mutation causes the disruption of a stabilizing H-bond pattern at the OM interface with receptors. G120A mutation equally impaired activations of ERK and STAT signaling pathways also by impairing the OM/cognate protein interactions. Interestingly, mutations of Gln20 and Asn123 differentially affected OM signaling through the two pathways. Q20A and N123A retained strong activity in inducing ERK phosphorylation but they showed diminished ability in activating STAT1 and STAT3. We further showed that mutations at Gln20 and Asn123 reduced OM induction of inflammatory gene fibrinogen-beta to a greater extent than that of LDL receptor gene. The mutation of Asn123 is directly related to local structural modification at site 3 of OM. Collectively these results provide a structural basis of OM-mediated signaling and suggest a potential to improve OM therapeutic properties via structural modification.

Peptide aptamers targeting mutant p53 induce apoptosis in tumor cells.

Mutations in the p53 tumor suppressor gene frequently result in expression of p53 point mutants that accumulate in cancer cells and actively collaborate with tumor progression through the acquisition of novel properties. Interfering with mutant p53 functions may represent a valid alternative for blocking tumor growth and development of aggressive phenotypes. The interactions and activities of selected proteins can be specifically modulated by the binding of peptide aptamers (PA). In the present work, we isolated PAs able to interact more efficiently with p53 conformational mutants compared with wild-type p53. The interaction between mutant p53 and PAs was further characterized using molecular modeling. Transient expression of PAs was able to reduce the transactivation activity of mutant p53 and to induce apoptosis specifically in cells expressing mutant p53. These PAs could provide a potential strategy to inhibit the oncogenic functions of mutant p53 and improve mutant p53-targeted cancer therapies.