Yubin Zhou

STIM1 gates the store-operated calcium channel ORAI1 in vitro

Store-operated Ca2+ entry through the plasma membrane Ca2+ release–activated Ca2+ (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca2+ flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.

Prediction of EF‐hand calcium‐binding proteins and analysis of bacterial EF‐hand proteins

The EF‐hand protein with a helix–loop–helix Ca2+ binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca2+ in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF‐hand and EF‐like Ca2+‐binding proteins. The canonical EF‐hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N‐ and C‐terminal EF‐hands within S100 and S100‐like proteins, a new signature profile has been established to allow for the identification of pseudo EF‐hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF‐hands. Furthermore, using the developed patterns, we have identified zero pseudo EF‐hand motif and 467 canonical EF‐hand Ca2+ binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF‐hand motifs are phylogenetically younger than canonical EF‐hand motifs. Our prediction of Ca2+ binding motifs provides not only an insight into the role of Ca2+ and Ca2+‐binding proteins in bacterial systems, but also a way to explore and define the role of Ca2+ in other biological systems (calciomics). Proteins 2006.

Initial activation of STIM1, the regulator of store-operated calcium entry

Physiological Ca2+ signaling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain was previously undefined. Using Tb3+-acceptor energy transfer, we show that dimerization of STIM1 ER-luminal domains causes an extensive conformational change in mouse STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane and communication with ORAI channels.

Multiple Ca(2+)-binding sites in the extracellular domain of the Ca(2+)-sensing receptor corresponding to cooperative Ca(2+) response.

A small change in the extracellular Ca(2+) concentration ([Ca(2+)](o)) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca(2+)-sensing receptors (CaSR). Mainly through binding of Ca(2+) to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca(2+) responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca(2+)-binding sites by grafting CaSR-derived, Ca(2+)-binding peptides to a scaffold protein, CD2, that does not bind Ca(2+). In this paper, we predict more potential noncontinuous Ca(2+)-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains two to three predicted Ca(2+)-binding sites. This approach enables us to further understand the mechanisms underlying the binding of multiple metal ions to extended polypeptides derived from a location within the ECD of the CaSR, which would be anticipated to more closely mimic the structure of the native CaSR ECD. Tb(3+) luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca(2+)-dependent conformational changes in these subdomains. Removing the predicted Ca(2+)-binding ligands in site 1 and site 3 abolishes the first binding step and second binding step, respectively. Studies on these subdomains suggest the existence of multiple metal-binding sites and metal-induced conformational changes that might be responsible for the switching on and off the CaSR by the transition between its open inactive form and closed active form.

Pore architecture of the ORAI1 store-operated calcium channel

ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca2+ signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca2+. We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca2+ binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter.

Viral calciomics: interplays between Ca2+ and virus.

Abstract Ca2+ is one of the most universal and versatile signaling molecules and is involved in almost every aspect of cellular processes. Viruses are adept at utilizing the universal Ca2+ signal to create a tailored cellular environment that meets their own demands. This review summarizes most of the known mechanisms by which viruses perturb Ca2+ homeostasis and utilize Ca2+ and cellular Ca2+-binding proteins to their benefit in their replication cycles. Ca2+ plays important roles in virion structure formation, virus entry, viral gene expression, posttranslational processing of viral proteins and virion maturation and release. As part of the review, we introduce an algorithm to identify linear “EF-hand” Ca2+-binding motifs which resulted in the prediction of a total of 93 previously unrecognized Ca2+-binding motifs in virus proteins. Many of these proteins are nonstructural proteins, a class of proteins among which Ca2+ interactions had not been formerly appreciated. The presence of linear Ca2+-binding motifs in viral proteins enlarges the spectrum of Ca2+–virus interplay and expands the total scenario of viral calciomics.

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor

Ca2+-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca2+ concentration ([Ca2+]o) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaSRs is the lack of adequate information about their Ca2+-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca2+-binding affinities. In this paper, we have reported the identification of three potential Ca2+-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca2+]o, similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca2+-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca2+-binding properties of the predicted sequences, we engineered two predicted continuous Ca2+-binding sequences individually into a scaffold protein provided by a non-Ca2+-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb3+ fluorescence energy transfer. Removing the predicted Ca2+-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca2+-binding residues were shown to be involved in Ca2+/Ln3+ binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca2+-binding sites within the extracellular domain of the CaSR.

Rational Design of Protein-based MRI Contrast Agents

We describe the rational design of a novel class of magnetic resonance imaging (MRI) contrast agents with engineered proteins (CAi.CD2, i = 1, 2,..., 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high-coordination Gd(3+) binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd(3+) over physiological metal ions such as Ca(2+), Zn(2+), and Mg(2+). These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small-molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, thereby extending applications of MRI.

Proteomic mapping of ER-PM junctions identifies STIMATE as a regulator of Ca2+ influx

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signalling. Store-operated Ca2+ entry mediated by dynamic STIM1–ORAI1 coupling represents a classical molecular event occurring at ER–PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry and a secondary screen based on bimolecular fluorescence complementation, we mapped the proteome of intact ER–PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER–PM junctions and suppresses the Ca2+–NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER–PM junctions.

Identification of the Calmodulin Binding Domain of Connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca2+-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136–158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca2+-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its α-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K+ is in the range of 0.7–1 μm. Upon binding of the peptide to CaM, the apparent Kd of Ca2+ for CaM decreased from 2.9 ± 0.1 to 1.6 ± 0.1 μm, and the Hill coefficient nH increased from 2.1 ± 0.1 to 3.3 ± 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca2+-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136–158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca2+-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca2+-dependent manner, providing a molecular basis for the well characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.