Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Logo for

Science 338 (6112): 1308-1313

Copyright © 2012 by the American Association for the Advancement of Science

Crystal Structure of the Calcium Release–Activated Calcium Channel Orai

Xiaowei Hou, Leanne Pedi, Melinda M. Diver, and Stephen B. Long*

Structural Biology Program, Memorial Sloan-Kettering Cancer Center (MSKCC), 1275 York Avenue, New York, NY 10065, USA.


Figure 1
View larger version (16K):
[in this window]
[in a new window]

 
Fig. 1. Channel reconstitution in liposomes. (A) Schematic of the fluorescence-based flux assay. Vesicles containing Orai or those prepared without protein (empty vesicles) were loaded with 150 mM NaCl and diluted 100-fold into flux buffer containing a fluorescent pH indicator (ACMA) and 150 mM N-methyl-D-glucamine (NMDG) to establish a Na+ gradient. After stabilization of the fluorescence signal (150 s), a proton ionophore (CCCP) was added to the sample, and an electrical potential arising from Na+ efflux was used to drive the uptake of protons into the vesicles, which quenches the fluorescence of ACMA. The "X" indicates that ACMA is no longer membrane-permeable in the protonated form. (B) Fluorescence measurements for the indicated protein constructs of Orai. Monensin, a Na+ ionophore, was added after 990 s to render all vesicles permeable to Na+ and establish a minimum fluorescence baseline. Fluorescence was normalized by dividing by the initial value. (C) Fluorescence trace observed for V174A Oraicryst in the absence and presence of Gd3+.

 

Figure 2
View larger version (28K):
[in this window]
[in a new window]

 
Fig. 2. Architecture of Orai. (A) Ribbon representation showing the tertiary structure of the channel from the side. The helices are colored: M1 (blue), M2 (red), M3 (green), M4 (brown), M4 extension (yellow in subunit A and gray in subunit B). Also shown are a Ca2+ ion (magenta sphere) and the nearby Glu178 residues (yellow sticks). Based on the hydrophobic region of the channel’s surface, horizontal lines (~30 Å apart) suggest approximate boundaries of the inner (In) and outer (Out) leaflets of the membrane. (B) Orthogonal view of the channel from the extracellular side. (C) Close-up view showing the interaction between the M4 extension helices.

 

Figure 3
View larger version (39K):
[in this window]
[in a new window]

 
Fig. 3. Ion pore. (A) Two M1 helices are drawn (four are omitted for clarity), showing the amino acids lining the pore in yellow. The approximate boundaries of the membrane-spanning region are shown as horizontal lines. The corresponding amino acids in human Orai1 are shown in parentheses. Ser161, Ser162, Thr164, Ser165, Ser169, and Gly170 are drawn as gray sticks. (B) View of the pore. Within a ribbon representation of four M1 helices (two in the foreground are removed for clarity) is a representation (teal) of the minimal radial distance from the center to the nearest van der Waals protein contact. The sections of the pore discussed in the text are labeled. (C and D) Molecular surface of Orai viewed from the extracellular (C) and intracellular (D) sides and colored according to the electrostatic potential contoured from –10 kT (red) to +10 kT (blue) (dielectric constant: 80).

 

Figure 4
View larger version (26K):
[in this window]
[in a new window]

 
Fig. 4. Cation binding in the external site. (A) Ba2+ and Ca2+ binding. The glutamate ring and hydrophobic section of the pore are depicted as in Fig. 3A. Anomalous-difference electron density for Ba2+ is shown in green mesh (calculated from 20 to 5 Å resolution using phases derived from the final model and contoured at 6{sigma} from a diffraction data set collected from a crystal soaked in 50 mM BaCl2 using x-rays with {lambda} = 1.70 Å. Electron density corresponding to Ca2+ is shown from a simulated annealing Fo-Fc omit map (blue mesh, calculated from 20 to 3.8 Å resolution and contoured at 4{sigma}) from a crystal soaked in 50 mM CaCl2 (table S1). The backbone carbonyl oxygen of Glu178 is also shown in stick representation. (B) Gd3+ binding. Anomalous-difference electron density is shown in purple mesh from a crystal soaked in 1 mM GdCl3 (calculated from 20 to 5 Å resolution using model phases and contoured at 11{sigma} from a diffraction data set collected using {lambda} = 1.70 Å x-rays).

 

Figure 5
View larger version (28K):
[in this window]
[in a new window]

 
Fig. 5. Anion binding and the K163W mutant. (A) Anomalous-difference electron density in the basic region of the WT pore contoured at 7{sigma} (orange mesh) and 14{sigma} (blue mesh) from a native unsoaked crystal. The M1 helices are depicted as in Fig. 3A. The map was calculated using phases derived from the protein model and a diffraction data set collected using {lambda} = 1.735 Å x-rays (resolution range 20 to 5 Å) (Table 1). (B) Anomalous-difference electron density present in the WT pore from a crystal soaked in (IrCl6)3–. The map is contoured at 7{sigma} (green mesh) and 20{sigma} (red mesh) (calculated using model phases from 20 to 5 Å resolution from a diffraction dataset collected using {lambda} = 1.1033 Å x-rays). (C and D) The K163W mutant. Two M1 helices of the K163W mutant are depicted in the same manner as in (A). (C) Electron density (cyan mesh) is shown for the tryptophan side chains at residue 163 (represented as sticks). The density is from a simulated annealing Fo-Fc map in which the tryptophan side chains have been removed from the model (calculated from 20 to 3.35 Å resolution and contoured at 3{sigma}). (D) Anomalous-difference electron density (green mesh) in the pore of a K163W mutant crystal soaked in (IrCl6)3– (calculated from 20 to 5 Å resolution using model phases and contoured at 7{sigma} from a diffraction data set collected using {lambda} = 1.1033 Å x-rays).

 

Figure 6
View larger version (35K):
[in this window]
[in a new window]

 
Fig. 6. Observed structure and hypothetical open state. (A) Observed (apparently closed) structure of Orai. The view is from the side with the M1 helices drawn as blue ribbons and the other helices shown as cylinders (M2, red; M3, green; M4 and M4 extension, yellow). A Ca2+ ion in the external site is depicted as a magenta sphere; an anion in the basic region of the pore is depicted as a gray sphere. Approximate boundaries of the lipid membrane are shown as horizontal lines. (B) Hypothetical model of an open state. The pore is widened by the outward dilation of the M1 helices (left-right arrow). A downward-pointing arrow indicates that Ca2+ is able to move though the pore unobstructed. The intracellular ends of the M1 helices are thought to interact with a cytosolic portion of STIM, as are the M4 extensions, which are modeled to protrude into the cytosol. The depiction of a cytosolic portion of STIM is meant to suggest that it might bridge the cytosolic portions of the M1 helices and the M4/M4 extension helices and is not meant to imply a particular structure, oligomeric state, or stoichiometry with Orai.

 


To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882