Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes.

• MeSH
• Autophagy * MeSH
• Cations, Divalent metabolism   MeSH
• Protein Conformation, alpha-Helical MeSH
• Humans MeSH
• EF Hand Motifs MeSH
• Mutation MeSH
• Myopathies, Structural, Congenital genetics   metabolism   MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Protein Unfolding MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

The stromal interaction molecule 1 (STIM1) has two important functions, Ca2+ sensing within the endoplasmic reticulum and activation of the store-operated Ca2+ channel Orai1, enabling plasma-membrane Ca2+ influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca2+-dependent conformations of the luminal STIM1 domain upon activation. Furthermore, we identified the residues within the canonical and noncanonical EF-hand domains that can bind to multiple Ca2+ ions. In MD simulations, a single Ca2+ ion was sufficient to stabilize the luminal STIM1 complex. Ca2+ store depletion destabilized the two EF hands, triggering disassembly of the hydrophobic cleft that they form together with the stable SAM domain. Point mutations associated with tubular aggregate myopathy or cancer that targeted the canonical EF hand, and the hydrophobic cleft yielded constitutively clustered STIM1, which was associated with activation of Ca2+ entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca2+ binding and refine the currently known initial steps of STIM1 activation on a molecular level.

• MeSH
• Algorithms MeSH
• Cell Membrane metabolism   MeSH
• Endoplasmic Reticulum metabolism   MeSH
• HEK293 Cells MeSH
• Hydrophobic and Hydrophilic Interactions MeSH
• Microscopy, Confocal MeSH
• Rats MeSH
• Humans MeSH
• EF Hand Motifs MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Neoplasm Proteins chemistry   genetics   metabolism   MeSH
• ORAI1 Protein chemistry   metabolism   MeSH
• Stromal Interaction Molecule 1 chemistry   genetics   metabolism   MeSH
• Protein Domains * MeSH
• Protein Unfolding * MeSH
• Molecular Dynamics Simulation * MeSH
• Calcium metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Trehalases hydrolyze the non-reducing disaccharide trehalose amassed by cells as a universal protectant and storage carbohydrate. Recently, it has been shown that the activity of neutral trehalase Nth1 from Saccharomyces cerevisiae is mediated by the 14-3-3 protein binding that modulates the structure of both the catalytic domain and the region containing the EF-hand-like motif, whose role in the activation of Nth1 is unclear. In this work, the structure of the Nth1·14-3-3 complex and the importance of the EF-hand-like motif were investigated using site-directed mutagenesis, hydrogen/deuterium exchange coupled to mass spectrometry, chemical cross-linking, and small angle x-ray scattering. The low resolution structural views of Nth1 alone and the Nth1·14-3-3 complex show that the 14-3-3 protein binding induces a significant structural rearrangement of the whole Nth1 molecule. The EF-hand-like motif-containing region forms a separate domain that interacts with both the 14-3-3 protein and the catalytic trehalase domain. The structural integrity of the EF-hand like motif is essential for the 14-3-3 protein-mediated activation of Nth1, and calcium binding, although not required for the activation, facilitates this process by affecting its structure. Our data suggest that the EF-hand like motif-containing domain functions as the intermediary through which the 14-3-3 protein modulates the function of the catalytic domain of Nth1.

• MeSH
• Enzyme Activation MeSH
• Catalytic Domain MeSH
• Models, Molecular MeSH
• EF Hand Motifs * MeSH
• 14-3-3 Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   metabolism   MeSH
• Saccharomyces cerevisiae enzymology   MeSH
• Amino Acid Sequence MeSH
• Trehalase chemistry   metabolism   MeSH
• Calcium metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.

• MeSH
• Ankyrin Repeat physiology   MeSH
• Transient Receptor Potential Channels chemistry   physiology   MeSH
• Humans MeSH
• Models, Molecular MeSH
• EF Hand Motifs physiology   MeSH
• Nerve Tissue Proteins chemistry   physiology   MeSH
• Molecular Dynamics Simulation MeSH
• Calcium metabolism   MeSH
• Calcium Channels chemistry   physiology   MeSH
• Binding Sites MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

An arsenic (ars) four-gene operon, containing genes encoding a putative membrane permease (ArsP), a transcriptional repressor (ArsR), an arsenate reductase (ArsC) and an arsenical-resistance membrane transporter (Acr3) was first identified in urease-positive thermophilic Campylobacter (UPTC) isolate, CF89-12. UPTC CF89-12 and some other Campylobacter lari isolates contained their ars four-genes, similarly, differing from that in the reference C. lari RM2100 strain. Two putative promoters and a putative terminator were identified for the operon in UPTC CF89-12. In vivo transcription of the operon was confirmed in the UPTC cells. PCR experiments using two primer pairs designed in silico to amplify two arsR and arsC-acr3 segments, respectively, generated two amplicons, approximately 200 and 350 base pairs, with all 31 of 31 and 19 of 31 C. lari isolates (n = 17 for UPTC; n = 14 for UN C. lari), respectively. An inverted repeat forming a dyad structure, a potential binding site for a transcriptional repressor, was identified in the promoter region. Within the deduced 61 amino acids sequence of the putative arsR open reading frame from the UPTC CF89-12, a metal binding box and a DNA-binding helix-turn-helix motif were identified. The UPTC CF89-12 and some other UPTC isolates isolated from natural environment were resistant to arsenate.

• MeSH
• Arsenic * MeSH
• Arsenate Reductases genetics   MeSH
• Genes, Bacterial * MeSH
• RNA, Bacterial genetics   MeSH
• Campylobacter lari genetics   isolation & purification   MeSH
• DNA, Bacterial genetics   MeSH
• DNA Primers MeSH
• Genetic Loci MeSH
• Nucleic Acid Conformation MeSH
• Molecular Sequence Data MeSH
• Helix-Turn-Helix Motifs genetics   MeSH
• Operon genetics   MeSH
• Open Reading Frames MeSH
• Reverse Transcriptase Polymerase Chain Reaction MeSH
• Amino Acid Sequence MeSH
• Sequence Analysis, DNA MeSH
• Sequence Alignment MeSH
• Urease genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• MeSH
• Research Support as Topic MeSH
• EF Hand Motifs genetics   MeSH
• Calcium-Binding Proteins analysis   MeSH
• Calcium Signaling physiology   MeSH
• Publication type
• Review MeSH