STIM1 (stromal interaction molecule 1) and Orai proteins are the essential components of Ca(2+) release-activated Ca(2+) (CRAC) channels. We focused on the role of cholesterol in the regulation of ...STIM1-mediated Orai1 currents. Chemically induced cholesterol depletion enhanced store-operated Ca(2+) entry (SOCE) and Orai1 currents. Furthermore, cholesterol depletion in mucosal-type mast cells augmented endogenous CRAC currents, which were associated with increased degranulation, a process that requires calcium influx. Single point mutations in the Orai1 amino terminus that would be expected to abolish cholesterol binding enhanced SOCE to a similar extent as did cholesterol depletion. The increase in Orai1 activity in cells expressing these cholesterol-binding-deficient mutants occurred without affecting the amount in the plasma membrane or the coupling of STIM1 to Orai1. We detected cholesterol binding to an Orai1 amino-terminal fragment in vitro and to full-length Orai1 in cells. Thus, our data showed that Orai1 senses the amount of cholesterol in the plasma membrane and that the interaction of Orai1 with cholesterol inhibits its activity, thereby limiting SOCE.
The channel Orai1 requires Ca
store depletion in the endoplasmic reticulum and an interaction with the Ca
sensor STIM1 to mediate Ca
signaling. Alterations in Orai1-mediated Ca
influx have been ...linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca
influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca
permeation associated with gene transcription and provide a gating mechanism for Orai1.
Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and ...missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein–protein and protein–DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The Ca(2+) release-activated Ca(2+) channel mediates Ca(2+) influx in a plethora of cell types, thereby controlling diverse cellular functions. The channel complex is composed of stromal interaction ...molecule 1 (STIM1), an endoplasmic reticulum Ca(2+)-sensing protein, and Orai1, a plasma membrane Ca(2+) channel. Channels composed of STIM1 and Orai1 mediate Ca(2+) influx even at low extracellular Ca(2+) concentrations. We investigated whether the activity of Orai1 adapted to different environmental Ca(2+) concentrations. We used homology modeling and molecular dynamics simulations to predict the presence of an extracellular Ca(2+)-accumulating region (CAR) at the pore entrance of Orai1. Furthermore, simulations of Orai1 proteins with mutations in CAR, along with live-cell experiments, or simulations and electrophysiological recordings of the channel with transient, electrostatic loop3 interacting with loop1 (the site of CAR) determined that CAR enhanced Ca(2+) permeation most efficiently at low external Ca(2+) concentrations. Consistent with these results, cells expressing Orai1 CAR mutants exhibited impaired gene expression stimulated by the Ca(2+)-activated transcription factor nuclear factor of activated T cells (NFAT). We propose that the Orai1 channel architecture with a close proximity of CAR to the selectivity filter, which enables Ca(2+)-selective ion permeation, enhances the local extracellular Ca(2+) concentration to maintain Ca(2+)-dependent gene regulation even in environments with relatively low Ca(2+)concentrations.
Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the ...Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73–91; Orai3 amino acids 48–65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
β-N-Acetylhexosaminidase (GH20) from the filamentous fungus Talaromyces flavus, previously identified as a prominent enzyme in the biosynthesis of modified glycosides, lacks a high resolution ...three-dimensional structure so far. Despite of high sequence identity to previously reported Aspergillus oryzae and Penicilluim oxalicum β-N-acetylhexosaminidases, this enzyme tolerates significantly better substrate modification. Understanding of key structural features, prediction of effective mutants and potential substrate characteristics prior to their synthesis are of general interest.
Computational methods including homology modeling and molecular dynamics simulations were applied to shad light on the structure-activity relationship in the enzyme. Primary sequence analysis revealed some variable regions able to influence difference in substrate affinity of hexosaminidases. Moreover, docking in combination with consequent molecular dynamics simulations of C-6 modified glycosides enabled us to identify the structural features required for accommodation and processing of these bulky substrates in the active site of hexosaminidase from T. flavus. To access the reliability of predictions on basis of the reported model, all results were confronted with available experimental data that demonstrated the principal correctness of the predictions as well as the model.
The main variable regions in β-N-acetylhexosaminidases determining difference in modified substrate affinity are located close to the active site entrance and engage two loops. Differences in primary sequence and the spatial arrangement of these loops and their interplay with active site amino acids, reflected by interaction energies and dynamics, account for the different catalytic activity and substrate specificity of the various fungal and bacterial β-N-acetylhexosaminidases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Charybdotoxin, belonging to the group of so-called scorpion toxins, is a short peptide able to block many voltage-gated potassium channels, such as mKv1.3, with high affinity. We use a reliable ...homology model based on the high-resolution crystal structure of the 94% sequence identical homologue Kv1.2 for charybdotoxin docking followed by molecular dynamics simulations to investigate the mechanism and energetics of unbinding, tracing the behavior of the channel protein and charybdotoxin during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force is constructed from the umbrella sampling simulations and combined with K d and free energy values gained experimentally using the patch-clamp technique to study the free energy of binding at different ion concentrations and the mechanism of the charybdotoxin–mKv1.3 binding process. A possible charybdotoxin binding mechanism is deduced that includes an initial hydrophobic contact followed by stepwise electrostatic interactions and finally optimization of hydrogen bonds and salt bridges.
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IJS, KILJ, NUK, PNG, UL, UM
The vanilloid receptor transient receptor potential (TRP)V1, also known as VR1 is a member of the TRP channel family. These receptors share a significant sequence homology, a similar predicted ...structure with six transmembrane-spanning domains (S1-S6), a pore-forming region between S5 and S6, and the cytoplasmically oriented C- and N-terminal regions. Although structural/functional studies have identified some of the key amino acids influencing the gating of the TRPV1 ion channel, the possible contributions of terminal regions to vanilloid receptor function remain elusive. In the present study, C-terminal truncations of rat TRPV1 have been constructed to characterize the contribution of the cytoplasmic C-terminal region to TRPV1 function and to delineate the minimum amount of C tail necessary to form a functional channel. The truncation of 31 residues was sufficient to induce changes in functional properties of TRPV1 channel. More pronounced effects of C-terminal truncation were seen in mutants lacking the final 72 aa. These changes were characterized by a decline of capsaicin-, pH-, and heat-sensitivity; progressive reduction of the activation thermal threshold (from 41.5 to 28.6 degrees C); and slowing of the activation rate of heat-evoked membrane currents (Q10 from 25.6 to 4.7). The voltage-induced currents of the truncated mutants exhibited a slower onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1 C-terminal domain (155 residues) resulted in a nonfunctional channel. These results indicate that the cytoplasmic COOH-terminal domain strongly influences the TRPV1 channel activity, and that the distal half of this structural domain confers specific thermal sensitivity.
The stromal interaction molecule 1 (STIM1) has two important functions, Ca
sensing within the endoplasmic reticulum and activation of the store-operated Ca
channel Orai1, enabling plasma-membrane Ca
...influx. We combined molecular dynamics (MD) simulations with live-cell recordings and determined the sequential Ca
-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 Ca
ions. In MD simulations, a single Ca
ion was sufficient to stabilize the luminal STIM1 complex. Ca
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 Ca
entry through Orai1 channels. On the basis of our results, we present a model of STIM1 Ca
binding and refine the currently known initial steps of STIM1 activation on a molecular level.
Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling ...of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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