Transient receptor potential vanilloid channel 3 (TRPV3), a member of the thermosensitive TRP (thermoTRPV) channels, is activated by warm temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. Mutations in trpv3 have been identified as the cause of the congenital skin disorder, Olmsted syndrome. Unlike other members of the thermoTRPV channel family, TRPV3 sensitizes upon repeated stimulation, yet a lack of structural information about the channel precludes a molecular-level understanding of TRPV3 sensitization and gating. Here, we present the cryo-electron microscopy structures of apo and sensitized human TRPV3, as well as several structures of TRPV3 in the presence of the common thermoTRPV agonist 2-aminoethoxydiphenyl borate (2-APB). Our results show α-to-π-helix transitions in the S6 during sensitization, and suggest a critical role for the S4-S5 linker π-helix during ligand-dependent gating.
Antisense oligonucleotides (AONs) can interfere with mRNA processing through RNase H-mediated degradation, translational arrest, or modulation of splicing. The antisense approach relies on AONs to efficiently bind to target sequences and depends on AON length, sequence content, secondary structure, thermodynamic properties, and target accessibility. We here performed a retrospective analysis of a series of 156 AONs (104 effective, 52 ineffective) previously designed and evaluated for splice modulation of the dystrophin transcript. This showed that the guanine-cytosine content and the binding energies of AON-target and AON-AON complexes were significantly higher for effective AONs. Effective AONs were also located significantly closer to the acceptor splice site (SS). All analyzed AONs are exon-internal and may act through steric hindrance of Ser-Arg-rich (SR) proteins to exonic splicing enhancer (ESE) sites. Indeed, effective AONs were significantly enriched for ESEs predicted by ESE software programs, except for predicted binding sites of SR protein Tra2beta, which were significantly enriched in ineffective AONs. These findings compile guidelines for development of AONs and provide more insight into the mechanism of antisense-mediated exon skipping. On the basis of only four parameters, we could correctly classify 79% of all AONs as effective or ineffective, suggesting these parameters can be used to more optimally design splice-modulating AONs.
Summary The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signaling. The mucolipin transient receptor potential (TRPML) channel family belongs to the TRP superfamily1,2 and is composed of three members, TRPML1-3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate organelle Ca2+ releases important for various physiological processes, including organelle trafficking and fusion3. Loss-of-function mutations in the TRPML1 gene cause the neurodegenerative lysosomal storage disorder mucolipidosis IV (ML-IV), and a gain-of-function mutation in TRPML3 (Ala419Pro) gives rise to the Varitint-Waddler (Va) mouse phenotype4–6. Notably, TRPMLs are activated by the low-abundance and LEL-enriched signaling lipid PI(3,5)P2, while other phosphoinositides such as PI(4,5)P2, enriched in plasma membranes, inhibit TRPMLs7,8. Conserved basic residues at the N-terminus of the channels are important for PI(3,5)P2 activation and PI(4,5)P2 inhibition8. However, due to a lack of structural information, the mechanism by which TRPML channels recognize PI(3,5)P2 and increase its Ca2+ conductance remains elusive. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3, at an average resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain we term the “mucolipin domain” (MLD). Combined with functional studies, we suggest that the MLD is responsible for PI(3,5)P2 binding and subsequent channel activation, and that it acts as a ‘gating pulley’ for lipid-dependent TRPML gating.
Summary Membrane transporters move substrates across the membrane by alternating access of their binding sites to one side of the membrane at a time. An emerging model is the elevator mechanism in which a substrate-binding transport domain moves a large distance across the membrane. This mechanism has been characterized by a transition between two states but the conformational path leading to the transition is not yet known, largely because the available structural information has been limited to the two end states. Here we present crystal structures of a concentrative nucleoside transporter from Neisseria wadsworthii representing inward-facing, intermediate, and outward-facing states. Interestingly, we determined the structures of multiple intermediate conformations in which the transport domain is captured halfway through its elevator motion. Our structures present a trajectory of the conformational transition in the elevator model, revealing multiple intermediate steps and state-dependent conformational changes within the transport domain associated with the elevator-type motion.
Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.It has long been hypothesized that mapping the spatial rearrangements that take place during the cycling between productive (active) and non-productive (inactive) states of kinases should lead to a better understanding of kinase dynamics, structure, function, and regulation (1-3). Furthermore, targeting kinase inhibitors to the inactive state is attractive because that form is more likely to represent a distinct conformation that may in turn lead to the identification of more selective inhibitors (4). However, despite advances in the field, designing inhibitors that target an inactive conformation of a kinase remains challenging (5) and largely empirical. The work described here provides a generally applicable methodology for the design of inhibitors that preferentially bind to the inactive state of a target kinase through developing a better understanding of the interplay between the conformational transitions that take place upon activation (6).Previous approaches have focused on the analysis of socalled "type II inhibitors" (7), which induce a distinct DFG-out conformation and occupy an additional hydrophobic pocket created by this rearrangement. The DFG-out conformation is a prerequisite for designing such type II inhibitors. The success of this strategy relies on the fact that despite the conserved nature of the ATP site, there are sufficient adjacent pockets that can ...
Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.
Human mitochondrial gene expression relies on the specific recognition and aminoacylation of mitochondrial tRNAs (mtRNAs) by nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs). Despite their essential role in cellular energy homeostasis, strong mutation pressure and genetic drift have led to an unparalleled sequence erosion of animal mtRNAs. The structural and functional consequences of this erosion are not understood. Here, we present cryo-EM structures of the human mitochondrial seryl-tRNA synthetase (mSerRS) in complex with mtRNASer(GCU). These structures reveal a unique mechanism of substrate recognition and aminoacylation. The mtRNASer(GCU) is highly degenerated, having lost the entire D-arm, tertiary core, and stable L-shaped fold that define canonical tRNAs. Instead, mtRNASer(GCU) evolved unique structural innovations, including a radically altered T-arm topology that serves as critical identity determinant in an unusual shape-selective readout mechanism by mSerRS. Our results provide a molecular framework to understand the principles of mito-nuclear co-evolution and specialized mechanisms of tRNA recognition in mammalian mitochondrial gene expression.
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