Crosslinking with BioATP-HDZ.

<p>(<b>A</b>) Structure of 2-azidoadenosine 5′-trisphosphate 2′,3′-biotin-long-chain-hydrazone (BioATP-HDZ). A highly reactive nitrene is produced by UV exposure and can form a covalent bond with a neighboring peptide backbone or amino acid side chain of the protein. (<b>B</b>) Fast-twitch skeletal muscle SR membranes were specifically labeled by 10 µM BioATP-HDZ and detected by IRDye800CW-streptavidin in-gel overlay. The left panel is the streptavidin in-gel overlay and the right panel is the CBB stain of the same gel. <i>Lane </i><i>1</i> – non-labeled SR membranes; <i>lane </i><i>2</i> – SR membranes labeled with BioATP-HDZ; <i>lane </i><i>3</i> – competition of BioATP-HDZ labeling by ATP in SR membranes. (<b>C</b>) Western blot with anti-RyR1 (pseudo-colored red) and anti-SERCA2 (pseudo-colored green) antibodies.</p

Identification of ATP-Binding Regions in the RyR1 Ca²⁺ Release Channel

<div><p>ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the <em>N</em>-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the <em>N</em>-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.</p> </div

Detection of BioATP-HDZ-labeled tryptic fragments.

<p>SR membranes were labeled with BioATP-HDZ in the absence (<b>A</b>, <b>B</b>) and presence (<b>C</b>, <b>D</b>) of ATP. Following the crosslinking, the SR membranes were digested with trypsin, solubilized with 2% CHAPS and separated by sucrose gradient centrifugation. Sucrose gradient fractions were analyzed by SDS-PAGE and labeled fragments were detected by in-gel IRDye800CW-streptavidin overlay at 800 nm. (<b>A</b>, <b>C</b>). The gels were then stained with CBB and scanned at 700 nm (<b>B</b>, <b>D</b>). Sucrose gradient RyR1 peak fractions are shown; fraction 20 was analyzed as an internal control for BioATP-HDZ labeling of SERCA. The number of the sucrose gradient fraction run in each lane is labeled above gels in A–D. (<b>E</b>) Purified RyR1 crosslinked with BioATP-HDZ, digested with trypsin and transferred to immobilon-FL membrane: <i>lane </i><i>1</i> – molecular weight standards; <i>lane </i><i>2</i> – IRDye800CW-streptavidin overlay; <i>lane </i><i>3</i> – CBB staining. (<b>F</b>) Immunoblotting of the trypsin-digested labeled RyR1 with a specific antibody against RyR1 amino acid sequence 416–434. (<b>G</b>) Plot of the normalized relative fluorescence (RFU) calculated for individual BioATP-HDZ-labeled tryptic fragments of RyR1 detected in SR membranes (blue) and purified RyR1 (red), error bars represent SEM (N = 3) and * indicates <i>p</i><0.03. The amount of BioATP-HDZ specifically bound with each labeled band in (<b>A</b>) is quantified using the equation [(<i>F</i>₈₀₀/<i>F</i>₇₀₀)_noATP – (<i>F</i>₈₀₀/<i>F</i>₇₀₀)_ATP], where [(<i>F</i>₈₀₀/<i>F</i>₇₀₀)_noATP is the total bound BioATP-HDZ measured as the fluorescence signal at 800 nm normalized to its respective CBB signal at 700 nm in (<b>B</b>). (<i>F</i>₈₀₀/<i>F</i>₇₀₀)_ATP estimates non-specific binding of BioATP-HDZ in (<b>C</b>) and (<b>D</b>). Calculations for SR membrane were performed using sucrose gradient fraction 7 with the exception of fragment 11, which was analyzed in fraction 20. The same quantifications were performed for the purified RyR1 shown in (<b>E</b>): <i>lane </i><i>2</i> – total bound BioATP-HDZ, <i>lane </i><i>3</i> – non-specific binding of BioATP-HDZ. Throughout the figure, bands detected by CBB stain and streptavidin overlay are numbered to the right of the gel by correspondence to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Zhang1" target="_blank">[37]</a>, bold numbers indicate BioATP-HDZ containing bands.</p

Map of BioATP-HDZ-labeled tryptic fragments of RyR1.

<p>(<b>A</b>) A linear schematic of RyR1 sequence; the black squares indicate the predicted transmembrane domains located at amino acids 3985–4937; major tryptic cleavage sites are indicated with vertical lines and corresponding amino acids are shown above. Arrows indicate locations of predicted conserved ATP-binding motifs in the primary structure of RyR1: solid arrows – Walker-A and the partial Walker-B motifs (amino acids 2–7, 699–704, 701–706, 1195–1200, 1302–1310, 2126–2234, 2264–2269, 2369–2377, 2370–2375, 4449–4452, 4452–4457 and 4602–4607), dashed arrows (amino acids 1081–1084, 2935–2938, 3503–3506 and 3937–3940) – the conserved RyR1 sequences similar to the ATP-binding site in GroES, grey arrows (amino acids 2369–2377, 2370–2375 and 3937–3940) – the motifs that were ruled out as ATP-binding sites in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Du2" target="_blank">[34]</a>, red arrows (amino acids 699–704, 701–706, 1081–1084 and 1195–1200) – motifs that can constitute ATP-binding sites in RyR1. Observed RyR1 tryptic fragments are depicted as rectangles: dashed lined – unlabeled fragments observed only in CBB-stained gels, green – BioATP-HDZ-labeled fragments, green-filled (amino acids 427–1302, 2402–2795 and 4476–5037) – labeled fragments that can potentially constitute ATP-binding sites. (<b>B</b>) Four predicted ATP-binding motifs found in fragment 8 (amino acids 427–1302).</p

Structural arrangement of RyR1 functional sites.

<p>(<b>A</b>) A linear schematic representation of the RyR1 amino acid sequence shows the three putative ATP-binding regions (green) with the predicted ATP-binding motifs (top panel). Positions for the peptides DP1 (amino acids 590–628) and DP4 (amino acids 2442–2477) are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Yamamoto1" target="_blank">[55]</a>. Several functional domains are represented in the linear sequence of RyR1 labeled in the bottom panel: MC/CCD – mutation hot-spot for malignant hyperthermia and central core disease, RIH – RyR/IP₃R homology regions, SPRY – SplA/RyR domain, LZ – leucine zippers, TM – transmembrane region. Sequences that were mapped in the 3D structure are indicated by the circled numbers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Jones1" target="_blank">[59]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Meng1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-PeralvezMarin1" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Liu4" target="_blank">[62]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Liu5" target="_blank">[72]</a>. (<b>B</b>) Surface representation of 1 nm resolution cryo-EM density map of RyR1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Ludtke1" target="_blank">[63]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Serysheva3" target="_blank">[64]</a> is shown for the top and side views. Left panels – computational localization of 2XOA in the clamp region, right panels – computational localization of 2XOA in the central region. The <i>N</i>- and <i>C</i>-terminus of the crystal structure of the <i>N</i>-terminal region (PDB ID: 2XOA) are represented as cyan and red spheres. The italic numbers indicate segmented subregions for one putative subunit. Subregions 1 and 6 are points where the segmentation of individual RyR1 subunits is ambiguous <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Serysheva3" target="_blank">[64]</a>, suggesting two alternative separations where either subregions 1 and 6, or 1′ and 6′ are included within the same subunit along with the remaining subregions. Circled numbers represent the location of the corresponding sequence in (<b>A</b>) mapped in the 3D structure. For technical reasons, localizations via GFP insertions were performed in RyR2, however due to the high sequence conservation between the two isoforms and near identical 3D structures <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Sharma1" target="_blank">[73]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Sharma2" target="_blank">[74]</a> the results are directly transferrable to RyR1 and the analogous RyR1 sequences are shown.</p

Quantification of the binding affinity of BioATP-HDZ to RyR1.

<p>SR membranes were labeled with 10 µM BioATP-HDZ in the absence or presence of increasing concentrations of ATP. The crosslinking of BioATP-HDZ to RyR1 was determined by IRDye800CW-streptavidin in-gel overlay (pseudo-colored green, <b>A</b>), and the signal was normalized to its respective CBB stain intensity at 700 nm (pseudo-colored red, <b>B</b>). (<b>C</b>) Quantification of BioATP-HDZ crosslinking to RyR1 as the mean of 3 independent experiments ± SEM. The IC₅₀ determined by non-linear regression was 0.6±0.2 mM.</p

Labeled fragments of RyR1 proteolytic complex.

a<p>– an apparent molecular weight as determined by mobility in SDS-PAGE.</p>b<p>– molecular weight of tryptic fragment calculated based on predicted trypsin cleavage sites in RyR1 sequence (Swiss-Prot accession number P11716).</p>c<p>– sequence not determined (ND) due to <i>N</i>-terminal blockage.</p>d<p>– SERCA sequence (Swiss-Prot accession number P04191).</p>e<p>– Calsequestrin 1 sequence (Swiss-Prot accession number P07221).</p>f<p>– non-identified sequence.</p

Sequence alignment of the putative ATP-binding regions in the RyR channel family.

<p>The aligned sequences are: rabbit RyR1 (GI: 134134), rabbit RyR2 (GI: 308153559), human RyR3 (GI: 126032338). Predicted secondary structure elements are given above the sequences and annotated as following: ‘e’ – β-strand, ‘h’ – α-helix; ‘c’ – coil; consensus sequences are shown below the aligned sequences with following symbols: upper-case – identical residues, lower-case – similar residues, dash – different residues. The putative ATP-binding sequences are highlighted with blue. (<b>A</b>) The ATP-binding motifs identified in the tryptic fragment 8: red – conserved glycine; purple – amino acid residues conforming to the ATP-binding motif in GroES. (<b>B</b>) The <i>N</i>-terminal sequence of fragment 15 aligned with the putative ATP-binding site in RyR2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048725#pone.0048725-Blayney1" target="_blank">[54]</a> are shown in orange.</p

Distribution profiles of SR membrane proteins across sucrose gradients.

<p>Amount of protein estimated by absorbance at 595 nm using BioRad protein assay reagent is plotted for each fraction collected from the sucrose gradients. Experimental conditions: blue line – digested SR membranes, red line – digested SR membranes in the presence of ATP, green line – digested SR membranes crosslinked with BioATP-HDZ, black line – digested SR membranes crosslinked with BioATP-HDZ in the presence of ATP. Western blot analyses of sucrose gradient fractions with anti-RyR1 antibody detected at 700 nm (right axis): dotted line – undigested SR membranes, dashed line – digested SR membranes.</p