Structural visualization of the p53/RNA polymerase II assembly.

Ryanodine-sensitive Ca2+ release channels, also known as the ryanodine receptors (RyRs), 2The abbreviations used are: RyR, ryanodine receptor; cryo-EM, cryoelectron microscopy; CY, cytoplasmic; TM, transmembrane; aa, amino acids; GFP, green fluorescent protein; IP3, inositol trisphosphate; CaM, calmodulin; FKBP, FK506-binding protein. are homotetramers of an ∼550-kDa subunit (Fig. 1) that are resident proteins of intracellular membranes such as the sarcoplasmic/endoplasmic reticulum. RyRs are responsible for the regulated release of Ca2+ from these lumenal stores. Over the last 10 years, single-particle cryoelectron microscopy (cryo-EM) has produced several low resolution (∼30 Å) structures of RyRs (for review, see Ref. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar). Recently, cryo-EM reconstructions of RyR1 have reached subnanometer resolution (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), a breakthrough due to improvements in cryo-specimen preparation, instrumentation, image processing, and three-dimensional reconstruction techniques. RyRs have a mushroom shape with 4-fold symmetry (Fig. 1B) (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Most of the mass of RyR forms a large cytoplasmic (CY) assembly (280 × 280 × 120 Å) that is connected to the transmembrane (TM) region by a stalk-like structure. The CY region is strikingly empty with numerous distinctive structural domains and intervening cavities that appear suitable for interaction with modulators that bind within the N-terminal regions of RyR (Fig. 1). The clamp-shaped regions, located at the corners of the CY assembly, are likely regions for the interdigitation of neighboring RyRs seen in situ (5Block B.A. Franzini-Armstrong C. J. Cell Biol. 1988; 107: 1099-1112Crossref PubMed Scopus (47) Google Scholar) or for interaction with modulators. The clamp-shaped regions are interconnected to form a continuous network between the central rim and the CY stalk-like structure via several bridging densities. The TM region (120 × 120 × 60 Å) is rotated by ∼40° with respect to the CY region. The pore is thought to be composed of six to eight TM segments (RyR1 TM1–8, amino acids (aa) 4277–4323, 4343–4363, 4557–4576, 4637–4662, 4776–4800, 4803–4825, 4834–4854, and 4911–4935 with aa 4854–4911 forming the pore helix and selectivity filter) (Fig. 1A, line 1) (6Du G.G. Sandhu B. Khanna V.K. Guo X.H. MacLennan D.H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16725-16730Crossref PubMed Scopus (118) Google Scholar). This arrangement places both the N and C termini of RyR in the cytoplasm. Sequence numbers used in this review refer to rabbit RyR1 (Swiss-Prot accession number P11716) and, where indicated, RyR2 (Swiss-Prot accession number P30957). The best current maps of RyR1 have resolutions of ∼10 Å (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar), allowing delineation of secondary structure elements in the pore region (Fig. 2A): the helix running across the membrane (helix 1, possibly TM8), the short helix (helix 2, which is likely to be part of the pore helix) that forms the lumenal entrance of the channel, and helix 3 (located parallel to the membrane plane on the CY side of the TM region). Helices 1 and 2 lie near the 4-fold channel axis and resemble the inner (pore-lining) and pore helices in K+ channel structures (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar, 8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). The orientation of helix 3 is similar to the slide helix seen in the KirBac1.1 structure (9Kuo A. Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Sciences (N. Y.). 2003; 300: 1922-1926Crossref PubMed Scopus (738) Google Scholar). Two recent RyR1 structures (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) were determined under conditions favoring the closed channel, the arrangement of the membrane-spanning helix that, in these structures, is quite different. Ludtke et al. (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) found a bent pore-lining helix (Fig. 2A) and suggested that the structure is similar to the open MthK channel (8Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1209) Google Scholar). This raises questions as to whether "kinking" of the inner helix alone, as proposed for K+ channels, is adequate to open RyRs. However, the conformation of the pore-lining helix in the other high resolution RyR1 cryo-EM structure (3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar) is straight and resembles the closed KcsA channel structure (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Sciences (N. Y.). 1998; 280: 69-77Crossref PubMed Scopus (5768) Google Scholar). These differences may due to resolution or to the conformational state of the channel during the imaging. At the current level of resolution in the three-dimensional structure of RyR, it is not possible to elucidate the number of TM helices. Single-particle cryo-EM has been used to generate structures of RyR1 in different conformational states to explore the structural transitions associated with the RyR gating. RyR1 was frozen under conditions determined from electrophysiological and biochemical studies (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) to drive the channel into either an opened or a closed state. Although the resolution of these structures was only 25–30 Å, global conformational changes associated with the closed-open transition of the RyR channel were detected in both the CY region (opening of the clamps) and the TM region (mass depletion from the center and a twisting motion of the TM region). Channel opening was proposed to be similar to the opening-closing of the iris in a camera diaphragm. Four approaches are used to identify the locations of specific sequences in the three-dimensional structures: 1) difference mapping with and without bound modulators; 2) difference mapping with and without sequence-specific antibodies; 3) insertion of tags such as green fluorescent protein (GFP) into defined positions in the primary sequence, followed by difference mapping; and 4) docking of molecular models of domains derived from crystal structures into the three-dimensional maps. There are three known RyR isoforms encoded by three distinct genes: RyR1 (expressed primarily in skeletal muscle), RyR2 (expressed in cardiac muscle and brain), and RyR3 (expressed in diaphragm, smooth muscle, and brain). These isoforms share ∼65% sequence identity. Regions of divergence (D1, aa 4254–4631 in RyR1 and aa 4210–4562 in RyR2; D2, aa 1342–1403 in RyR1 and aa 1353–1397 in RyR2; and D3, aa 1872–1923 in RyR1 and aa 1852–1890 in RyR2) are indicated in Fig. 1A (line 2). These divergent regions could be responsible for important isoform-specific functions, or they could represent regions that are not evolutionarily conserved. Three-dimensional structures of all three RyR isoforms determined at ∼30 Å using single-particle cryo-EM are virtually identical (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Sharma M.R. Jeyakumar L.H. Fleischer S. Wagenknecht T. J. Biol. Chem. 2000; 275: 9485-9491Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Using GFP insertions, D1 was mapped to subregion 3, whereas both D2 and D3 were mapped to the clamp in subregions 6 and 9, respectively (Fig. 1B). The three divergent regions all appear to be located in the CY domains close to the top of the structure, as would be expected for regions of RyR1 involved in interactions with CaV1.1. However, whether they are involved remains to be determined. Analysis of the primary sequence of RyR1 has revealed several motifs found in other proteins, but the functional roles of these motifs in RyRs are unknown. These motifs include 1) three SPRY domains (aa 582–798, 1014–1209, and 1358–1571) (Fig. 1A, line 3), which are domains found in members of the immunoglobulin superfamily; 2) a sequence that is repeated four times in two tandem domains (aa 842–955, 956–1069, 2726–2845, and 2846–2959) (Fig. 1A, line 4) with two additional truncated repeats located between the two domains (aa 1345–1360 and 1373–1388); 3) five MIR domains (aa 98–153, 160–205, 211–265, 271–334, and 336–394) (Fig. 1A, line 5), which are sequences found in RyRs, inositol trisphosphate receptors, and protein O-mannosyltransferases; and 4) two or more leucine zippers (aa 554–575 and 3039–3071 in RyR1 and aa 565–594, 1604–1632, and 3005–3036 in RyR2) (Fig. 1A, line 6), which are protein-protein interaction motifs and have been suggested to anchor PP1/spinophilin, PP2A/PR130, and protein kinase A/RII (protein kinase A regulatory subunit II)/mAKAP (muscle A-kinase anchoring protein) to RyR2 (13Marks A.R. Marx S.O. Reiken S. Trends Cardiovasc. Med. 2002; 12: 166-170Crossref PubMed Scopus (79) Google Scholar). The MIR domains are all located close to the N terminus, which is part of the clamp domain, suggesting that these domains may contribute to the structure/function of the clamps (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Computational placement of a homology model based on the crystal structure of the inositol trisphosphate (IP3)-binding and suppressor domains of the IP3 receptor (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) places the first leucine zipper (aa 554–574) in the clamp domain in subregion 7 (Figs. 1B and 2B). If this is indeed the site that anchors kinases or phosphatases to RyR1, the targets of the enzymes may be sequences in close proximity to this site. Stewart et al. (15Stewart R. Zissimopoulos S. Lai F.A. Biochem. J. 2003; 376: 795-799Crossref PubMed Scopus (36) Google Scholar) proposed that the last 15 amino acids in the C-terminal tail are required for tetramer formation. Galvan and Mignery (16Galvan D.L. Mignery G.A. J. Biol. Chem. 2002; 277: 48248-48260Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) also suggested a C-terminal tetramerization domain but suggested that this was between aa 4869 and 5019 in RyR1. These regions are indicated in Fig. 1A (line 7). Because the C termini of RyRs are cytoplasmic, the region of tetramerization must also be in CY domains. The activities of all three RyR isoforms are modulated by a large number of agents, including ATP, Ca2+, Mg2+, calmodulin, FKBP12, junctin, triadin, calsequestrin, S100A, sorcin, and many others. Most of these modulators bind to the CY domains of the RyRs and allosterically regulate the opening of the Ca2+ conduction pathway in the TM region. RyR activities are also modulated by post-translation modifications such as phosphorylation, oxidation, and S-nitrosylation. Prerequisites for the delineation of the mechanisms of channel regulation by individual modulators and modifications are the identity of the sites of modulator interaction or modification in the primary sequence and the location of these sites in the three-dimensional structure. Single-particle cryo-EM has been used to analyze macromolecular interactions of RyR with the modulatory proteins calmodulin and FKBP12 (for original references on these studies, see reviews in Refs. 1Serysheva I.I. Chiu W. Ludtke S.J. Methods Cell Biol. 2007; 79: 407-435Crossref PubMed Scopus (8) Google Scholar and 17Durham W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New York Inc., New York2007: 273-321Google Scholar). Because most channel modulators bind in the CY assembly, there must be mechanisms whereby changes in the conformation of specific domains in the CY assembly are transduced, first into changes in the column-like structure and then into changes in the TM helices of the pore. CaV1.1—In skeletal muscle, the interaction between CaV1.1 in the transverse tubules and RyR1 in the sarcoplasmic reticulum has been shown to involve an orthograde signal, whereby CaV1.1 signals RyR1 to open, and a retrograde signal, through which RyR1 increases the Ca2+ current through CaV1.1 (18Dirksen R.T. Front. Biosci. 2002; 7: d659-d670Crossref PubMed Google Scholar). Multiple regions of RyR1 are likely to be involved in the coupling to CaV1.1 (Fig. 1A, line 8) (reviewed in Ref. 18Dirksen R.T. Front. Biosci. 2002; 7: d659-d670Crossref PubMed Google Scholar), including aa 1635–2636, 2659–3720, and 1837–2154 and multiple regions between aa 1 and 1680 (19Sheridan D.C. Takekura H. Franzini-Armstrong C. Beam K.G. Allen P.D. Perez C.F. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 19760-19765Crossref PubMed Scopus (62) Google Scholar). The sites of interaction appear to be distributed over a rather large part of the CY domain of RyR1. Based on freeze-fracture studies (20Paolini C. Fessenden J.D. Pessah I.N. Franzini-Armstrong C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12748-12752Crossref PubMed Scopus (63) Google Scholar), the most likely regions of RyR1 to be in contact with CaV1.1 include subregions 4 and 6 (Fig. 1B). Several of the sequences suggested to contribute to mechanical coupling have been found within these two subdomains. Ser437 of RyR2 (Ser422 in RyR1) maps to a location between subregions 5 and 9. Ser2368 in RyR2 (Arg2400 in RyR1) maps to a location between subregions 5 and 6. An interaction of CaV1.1 with these regions of RyR1 could directly control the opening of the clamp and allosterically modulate the channel gating. Ca2+—Regulation of RyRs by Ca2+ varies with the RyR isoform. The most important modulator of RyR2 activity in cardiac muscle is Ca2+, where Ca2+ enters through CaV1.2 and activates RyR2. Ca2+ also regulates RyR1 activity, but depending on the concentration, it can either activate or inhibit its activity. These findings suggest that RyR1 has at least two Ca2+-binding sites. Both RyR1 and RyR2 are activated at 0.1–10 μm Ca2+ (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), but RyR1 is more sensitive to inhibition by higher concentrations of Ca2+. Lumenal Ca2+ also regulates the activity of RyRs (22Jiang D. Xiao B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13062-13067Crossref PubMed Scopus (361) Google Scholar). Possible binding sites for Ca2+ (Fig. 1A, line 9) are located between aa 1861 and 2094 (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), 3657 and 3776 (21Chen S.R. MacLennan D.H. J. Biol. Chem. 1994; 269: 22698-22704Abstract Full Text PDF PubMed Google Scholar), and 4381 and 4626 (23Treves S. Chiozzi P. Zorzato F. Biochem. J. 1993; 291: 757-763Crossref PubMed Scopus (26) Google Scholar) and two putative EF-hand motifs located at aa 4079–4092 (EF1) and 4115–4126 (EF2) (24Xiong H. Feng X. Gao L. Xu L. Pasek D.A. Seok J.H. Meissner G. Biochemistry. 1998; 37: 4804-4814Crossref PubMed Scopus (54) Google Scholar). Mutation E3986A (Glu4042 in rabbit RyR1) in mouse RyR2 reduced Ca2+ activation by >1000-fold (25Li P. Chen S.R. J. Gen. Physiol. 2001; 118: 33-44Crossref PubMed Scopus (113) Google Scholar). Fessenden et al. (26Fessenden J.D. Feng W. Pessah I.N. Allen P.D. J. Biol. Chem. 2004; 279: 53028-53035Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) scrambled the putative EF-hand motifs and found that these mutations did not alter the response to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) in intact myotubes. Surprisingly, no high affinity [3H]ryanodine binding was observed in membranes expressing the EF2 mutation, but the channels in bilayers were activated by Ca2+ in the micromolar range. These findings suggest that the putative EF-hands contribute to Ca2+ modulation of the channel but are not sufficient for Ca2+ regulation of this close to these putative Ca2+-binding sites have not been mapped in the three-dimensional structures of RyRs. However, et al. L. Zhang R. Hamilton S.L. Biophys. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) suggested an interaction of the regions putative and EF2 with the calmodulin region (aa suggesting that these two EF-hands are close to 3, where the site is located also bind CaM, which is a Ca2+-binding protein. RyR1 is the protein of sarcoplasmic reticulum membranes A. Xu L. G. Meissner G. Biophys. J. Full Text PDF PubMed Scopus Google Scholar). At micromolar Ca2+ both RyR1 and RyR2. However, at activates RyR1 but RyR2 D. and A. in Scholar). RyR3 also CaM, and the of on the channel are Xu L. Pasek D.A. Chen S.R. Meissner G. Biochemistry. 2005; PubMed Scopus Google Scholar). The site on RyR1 aa (Fig. 1A, line G. Zhang L. G. Hamilton S.L. Biochemistry. 1999; PubMed Scopus Google Scholar, Xu L. Pasek D.A. Meissner G. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) and at an close to aa H. Zhang Hamilton S.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). difference the site was found to be located in CY subregion 3 (Fig. 1B). the location of the site is to Å in the of Ca2+ with respect to its for M. Wagenknecht T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). This of could be due to a of binding Ca2+ a of the site RyR1 Ca2+. These findings suggest that subregion 3 a regulatory for channel activation and but additional studies are required to elucidate the molecular of regulation of RyR1. FKBP12 and bind to RyRs and regulate activities A.R. Physiol. 1996; PubMed Scopus Google Scholar). Although FKBP12 and are to bind to all RyR FKBP12 with RyR1, whereas with RyR2. The bound to RyR3 is not bind to RyR1 and RyR2 on site RyR1 subunit and a closed state of the channel A.R. Physiol. 1996; PubMed Scopus Google Scholar). The binding site has been suggested to be between aa and in RyR1 (Fig. 1A, line Mutation of this site FKBP12 binding M. J. Reiken S. F. Marx S.O. A.R. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), but this may be due to an regulation of the binding site S. Lai F.A. Cell Biochem. Biophys. 2005; PubMed Scopus Google Scholar). Using difference mapping of three-dimensional reconstructions of RyR1 with and without FKBP12, the binding site for FKBP12 is found at the between clamp subregions 5 and and subregion 3, neighboring clamps in the RyR CY region (Fig. 1B). of the location of FKBP12 by difference mapping M. X. Allen P.D. J. Mol. Biol. 2006; PubMed Scopus Google Scholar) with the docking of the IP3 homology model (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar) that there is a binding for FKBP12 by and (Fig. 2B). or not FKBP12 at this remains to be protein has been shown to with RyR1 and to regulate its response to both and W. J. Yang T. Allen P.D. Pessah I.N. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (125) Google Scholar). A possible sequence is found between aa and has been mapped between domains 5 and 6 Wang R. Zhang J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that may also via the clamp domain of RyR1. and S. A. H. A. C. F. M. J. Wang J. D. G. A.R. J. Cell Biol. 2003; PubMed Scopus Google Scholar) have suggested that RyR (aa in RyR1 and aa in RyR2) from RyRs, to Ca2+ A number of other have to these findings 2007; 101: PubMed Scopus Google Scholar, S. J. R. 2006; PubMed Scopus Google Scholar, G. H. Chen X. H. S.R. 2008; PubMed Scopus Google Scholar). site of is aa (aa in Possible sites are indicated in Fig. 1A (line of GFP at in RyR2 in RyR1) this site to domain to the site X. Xiao B. S. X. F. J. R. J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The putative site was mapped by GFP at and was found to be located in to the site X. Xiao B. S. J. Wagenknecht T. Chen S.R. Biochem. J. 2008; PubMed Scopus Google Scholar). are in the is the primary of (Fig. 1, line in J. C. Meissner G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). et al. W.J. P. C. S. Galvan D.L. Baker M.R. F. R. Hamilton S.L. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar) that RyR1 the found with RyR1 mutations associated with have been by et al. P. R.T. C. Hamilton S.L. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was primarily in a open conformation (Fig. 1A, line and by et al. J. M. D. Pessah I.N. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar) under conditions in which the channel was in a primarily closed Although the differences may be due to differences in it is also possible that different are the channel is in the open closed are the in the three-dimensional Several can be in the docking model (Fig. is in the site and is located in are located in 5 and 6 (Fig. 1B) the region from aa to to these Wang R. Zhang J. Chen S.R. Wagenknecht T. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). in RyR1 central and of recent review by et al. W.J. Wehrens X.H.T. Sood S. Hamilton S.L. Carafoli E. Brini M. Calcium Signaling and Disease. Springer-Verlag New York Inc., New York2007: 273-321Google Although there is for the mutations to in three regions of RyR1, it that mutations can the primary sequence (Fig. 1A, line that the channel to be more sensitive to whereas central mutations either the channel or through the for the location of of the N-terminal mutations can be from the docking model (Fig. 2B). Channel opening is associated with structural transitions in the CY clamp subregions and (Fig. 2A) (10Orlova E.V. Serysheva I.I. van Heel M. Hamilton S.L. Chiu W. Nat. Struct. Biol. 1996; 3: 547-552Crossref PubMed Scopus (146) Google Scholar, 11Serysheva I.I. Schatz M. van Heel M. Chiu W. Hamilton S.L. Biophys. J. 1999; 77: 1936-1944Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). the three-dimensional locations of the and sites and other important functional the clamp subregions are likely to be involved in modulation of the channel activity. These findings suggest a between the conformation of the clamp and channel with many mutations associated with and central in mutations that the channel to open more are located in clamp subregions and (Fig. 2) (4Serysheva I.I. Ludtke S.J. Baker M.L. Cong Y. Topf M. Eramian D. Sali A. Hamilton S.L. Chiu W. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 9610-9615Crossref PubMed Scopus (95) Google Scholar). Although RyR1 was the first structures by single-particle cryo-EM at resolution M. R. J. Fleischer S. Wagenknecht T. J. Cell Biol. 1994; PubMed Scopus Google Scholar, I.I. E.V. Chiu W. Hamilton S.L. van Heel M. Nat. Struct. Biol. PubMed Scopus Google Scholar), the RyR assembly remains the most for structural due to its RyR1 reconstructions at ∼10 Å by two have important into its pore structure, and control by multiple intracellular (2Ludtke S.J. Serysheva I.I. Hamilton S.L. Chiu W. Structure (Camb.). 2005; 13: 1203-1211Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 3Samso M. Wagenknecht T. Allen P.D. Nat. Struct. Mol. Biol. 2005; 12: 539-544Crossref PubMed Scopus (168) Google Scholar). Several higher resolution structures of RyR1, including from cryo-specimen of membrane proteins, of the protein from its with and of different conformational are to the channel in a functional state and either to all modulators or to all binding sites with modulators. New are to the number of has to be and the of are that of these to be are 1) crystal structures of individual or into the cryo-EM and 2) and the models into the cryo-EM the in the resolution of RyR structure are likely to in the with L. Serysheva

Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.

Tetramerisation of α-latrotoxin by divalent cations is responsible for toxin-induced non-vesicular release and contributes to the Ca 2+-dependent vesicular exocytosis from synaptosomes

The replication and packaging of the rotavirus genome, comprising 11 segments of double-stranded RNA, take place in specialized compartments called viroplasms, which are formed during infection and involve a coordinated interplay of multiple components. Two rotavirus nonstructural proteins, NSP2 (with nucleoside triphosphatase, single-stranded RNA [ssRNA] binding and helix-destabilizing activities) and NSP5, are essential in these events. Previous structural analysis of NSP2 showed that it is an octamer in crystals, obeying 4-2-2 crystal symmetry, with a large 35-A central hole along the fourfold axis and deep grooves at one of the twofold axes. To ascertain that the solution structure of NSP2 is the same as that in the crystals and investigate how NSP2 interacts with NSP5 and RNA, we carried out single-particle cryoelectron microscopy (cryo-EM) analysis of NSP2 alone and in complexes with NSP5 and ssRNA at subnanometer resolution. Because full-length NSP5 caused severe aggregation upon mixing with NSP2, the deletion construct NSP566-188 was used in these studies. Our studies show that the solution structure of NSP2 is same as the crystallographic octamer and that both NSP566-188 and ssRNA bind to the grooves in the octamer, which are lined by positively charged residues. The fitting of the NSP2 crystal structure to cryo-EM reconstructions of the complexes indicates that, in contrast to the binding of NSP566-188, the binding of RNA induces noticeable conformational changes in the NSP2 octamer. Consistent with the observation that both NSP5 and RNA share the same binding site on the NSP2 octamer, filter binding assays showed that NSP5 competes with ssRNA binding, indicating that one of the functions of NSP5 is to regulate NSP2-RNA interactions during genome replication.

N-methyl-D-aspartate receptors (NMDARs) are critically involved in basic brain functions and neurodegeneration as well as tumor invasiveness. Targeting specific subtypes of NMDARs with distinct activities has been considered an effective therapeutic strategy for neurological disorders and diseases. However, complete elimination of off-target effects of small chemical compounds has been challenging and thus, there is a need to explore alternative strategies for targeting NMDAR subtypes. Here we report identification of a functional antibody that specifically targets the GluN1-GluN2B NMDAR subtype and allosterically down-regulates ion channel activity as assessed by electrophysiology. Through biochemical analysis, x-ray crystallography, single-particle electron cryomicroscopy, and molecular dynamics simulations, we show that this inhibitory antibody recognizes the amino terminal domain of the GluN2B subunit and increases the population of the non-active conformational state. The current study demonstrates that antibodies may serve as specific reagents to regulate NMDAR functions for basic research and therapeutic objectives.

Liver injury associated with exposure to therapeutic agents that undergo hepatic metabolism can involve the formation of reactive metabolites. These may cause redox perturbation which can result in oxidative stress as well as protein modification leading to activation or inhibition of cellular transcriptional responses. Nevertheless, the effects of these challenges on more than one transcriptional pathway simultaneously remain unclear. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-κB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins. We also employed RNAi to manipulate Keap1 (the inhibitor of Nrf2), Nrf2 itself and NF-κB-p65, to understand their roles in the response to drug stress. All three chemicals induced Nrf2, but NF-κB binding activity was only increased after BSO treatment. In fact, NF-κB binding activity decreased after exposure to NAPQI and DNCB. While RNAi depletion of Keap1 led to reduced toxicity following exposure to DNCB, depletion of Nrf2 and NF-κB augmented toxicity. Interestingly, increased Nrf2 caused by Keap1 depletion was reversed by co-depletion of NF-κB. We demonstrate that Keap1/Nrf2 and NF-κB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-κB to partly influence Nrf2 expression during cellular stress.

Background/Objectives: Acetaminophen (APAP) is used during 50–60% of pregnancies in the U.S. and has been associated with childhood respiratory morbidity, though the underlying mechanism remains unclear. APAP-induced injury is dependent on cell-specific expression of CYP2E1, the enzyme that metabolizes APAP into the mitochondrial toxin NAPQI. In mice, pulmonary Cyp2e1 expression peaks during the saccular stage of lung development on embryonic day 18 (E18). We investigated whether this developmental surge in Cyp2e1 triggers a pulmonary transcriptional response to maternal APAP exposure in embryonic lungs. Methods: Pregnant dams were exposed to APAP on E17 or E18 (150 or 250 mg/kg, IP) using doses derived from prior studies. We assessed the induction of NRF2 target genes and genes associated with inflammation, apoptosis and cellular stress due to their roles in APAP-induced oxidative and cellular stress. Results: At E17, maternal treatment with APAP induced pulmonary Cyp2e1 but resulted in inconsistent transcriptional changes. In contrast, maternal APAP at E18 triggered a robust transcriptional induction of Cyp2e1, NRF2 targets and markers of apoptosis, inflammation and cellular stress. Histopathology at birth after E18 APAP exposure revealed no acute pulmonary injury. Conclusions: We demonstrate a developmentally regulated, dose-dependent transcriptional response to maternal APAP in the embryonic murine lung. Importantly, transcriptional responses do not directly indicate lung injury; thus, future studies should assess protein-level changes following APAP exposure. This study underscores the need for further investigation into the role of developmentally regulated Cyp2e1 expression in APAP-induced toxicity and long-term respiratory morbidity.

BackgroundVaccination with radiation-attenuated Plasmodium falciparum sporozoites is known to induce protective immunity. However, the mechanisms underlying this protection remain unclear. In this work, two recent radiation-attenuated sporozoite vaccination studies were used to identify potential transcriptional correlates of vaccination-induced protection.MethodsLongitudinal whole blood RNAseq transcriptome responses to immunization with radiation-attenuated P. falciparum sporozoites were analysed and compared across malaria-naïve adult participants (IMRAS) and malaria-experienced adult participants (BSPZV1). Parasite dose and method of delivery differed between trials, and immunization regimens were designed to achieve incomplete protective efficacy. Observed protective efficacy was 55% in IMRAS and 20% in BSPZV1. Study vaccine dosings were chosen to elicit both protected and non-protected subjects, so that protection-associated responses could be identified.ResultsAnalysis of comparable time points up to 1 week after the first vaccination revealed a shared cross-study transcriptional response programme, despite large differences in number and magnitude of differentially expressed genes between trials. A time-dependent regulatory programme of coherent blood transcriptional modular responses was observed, involving induction of inflammatory responses 1–3 days post-vaccination, with cell cycle responses apparent by day 7 in protected individuals from both trials. Additionally, strongly increased induction of inflammation and interferon-associated responses was seen in non-protected IMRAS participants. All individuals, except for non-protected BSPZV1 participants, showed robust upregulation of cell-cycle associated transcriptional responses post vaccination.ConclusionsIn summary, despite stark differences between the two studies, including route of vaccination and status of malaria exposure, responses were identified that were associated with protection after PfRAS vaccination. These comprised a moderate early interferon response peaking 2 days post vaccination, followed by a later proliferative cell cycle response steadily increasing over the first 7 days post vaccination. Non-protection is associated with deviations from this model, observed in this study with over-induction of early interferon responses in IMRAS and failure to mount a cell cycle response in BSPZV1.

Ebola virus causes haemorrhagic fever with a high fatality rate in humans and non-human primates. It belongs to the family Filoviridae in the order Mononegavirales, which are viruses that contain linear, non-segmented, negative-sense, single-stranded genomic RNA1,2. The enveloped, filamentous virion contains the nucleocapsid, consisting of the helical nucleoprotein-RNA complex, VP24, VP30, VP35 and viral polymerase1,3. The nucleoprotein-RNA complex acts as a scaffold for nucleocapsid formation and as a template for RNA replication and transcription by condensing RNA into the virion4,5. RNA binding and nucleoprotein oligomerization are synergistic and do not readily occur independently6. Although recent cryo-electron tomography studies have revealed the overall architecture of the nucleocapsid core4,5, there has been no high-resolution reconstruction of the nucleocapsid. Here we report the structure of a recombinant Ebola virus nucleoprotein-RNA complex expressed in mammalian cells without chemical fixation, at near-atomic resolution using single-particle cryo-electron microscopy. Our structure reveals how the Ebola virus nucleocapsid core encapsidates its viral genome, its sequence-independent coordination with RNA by nucleoprotein, and the dynamic transition between the RNA-free and RNA-bound states. It provides direct structural evidence for the role of the N terminus of nucleoprotein in subunit oligomerization, and for the hydrophobic and electrostatic interactions thatlead to theformation of the helical assembly. The structure is validated as representative of the native biological assembly of the nucleocapsid core by consistent dimensions and symmetry with the full virion5. The atomic model provides a detailed mechanistic basis for understanding nucleocapsid assembly and highlights key structural features that may serve as targets for anti-viral drug development.

6-Nonadecynoic acid (6-NDA), a plant-derived acetylenic acid, exhibits strong inhibitory activity against the human fungal pathogens Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes. In the present study, transcriptional profiling coupled with mutant and biochemical analyses were conducted using the model yeast Saccharomyces cerevisiae to investigate its mechanism of action. 6-NDA elicited a transcriptome response indicative of fatty acid stress, altering the expression of genes that are required for yeast growth in the presence of oleate. Mutants of S. cerevisiae lacking transcription factors that regulate fatty acid β-oxidation showed increased sensitivity to 6-NDA. Fatty acid profile analysis indicated that 6-NDA inhibited the formation of fatty acids longer than 14 carbons in length. In addition, the growth inhibitory effect of 6-NDA was rescued in the presence of exogenously supplied oleate. To investigate the response of a pathogenic fungal species to 6-NDA, transcriptional profiling and biochemical analyses were also conducted in C. albicans. The transcriptional response and fatty acid profile of C. albicans were comparable to those obtained in S. cerevisiae, and the rescue of growth inhibition with exogenous oleate was also observed in C. albicans. In a fluconazole-resistant clinical isolate of C. albicans, a fungicidal effect was produced when fluconazole was combined with 6-NDA. In hyphal growth assays, 6-NDA inhibited the formation of long hyphal filaments in C. albicans. Collectively, our results indicate that the antifungal activity of 6-NDA is mediated by a disruption in fatty acid homeostasis and that 6-NDA has potential utility in the treatment of superficial Candida infections.

The S. cerevisiae SUMO stress response is a conjugation-deconjugation cycle that targets the transcription machinery.

Transcriptomic research provides a mechanistic understanding of an organism's response to environmental challenges such as increasing temperatures, which can provide key insights into the threats posed by thermal challenges associated with urbanization and climate change. Differential gene expression and alternative splicing are two elements of the transcriptomic stress response that may work in tandem, but relatively few studies have investigated these interactions in fishes of conservation concern. We studied the imperilled redside dace (Clinostomus elongatus) as thermal stress is hypothesized to be an important cause of population declines. We tested the hypothesis that gene expression-splicing interactions contribute to the thermal stress response. Wild fish exposed to acute thermal stress were compared with both handling controls and fish sampled directly from a river. Liver tissue was sampled to study the transcriptomic stress response. With a gene set enrichment analysis, we found that thermally stressed fish showed a transcriptional response related to transcription regulation and responses to unfolded proteins, and alternatively spliced genes related to gene expression regulation and metabolism. One splicing factor, prpf38b, was upregulated in the thermally stressed group compared with the other treatments. This splicing factor may have a role in the Jun/AP-1 cellular stress response, a pathway with wide-ranging and context-dependent effects. Given large gene interaction networks and the context-dependent nature of transcriptional responses, our results highlight the importance of understanding interactions between gene expression and splicing for understanding transcriptomic responses to thermal stress. Our results also reveal transcriptional pathways that can inform conservation breeding, translocation and reintroduction programs for redside dace and other imperilled species by identifying appropriate source populations.

Reactive oxygen species (ROS) are an important source of cellular damage. When ROS intracellular levels increase, oxidative stress takes place affecting DNA stability and metabolic functions. To prevent these effects, stress-activated protein kinases (SAPKs) delay cell cycle progression and induce a transcriptional response that activates antioxidant mechanisms ensuring cell adaptation and survival. Fission yeast Cdc14-like phosphatase Flp1 (also known as Clp1) has a well-established role in cell cycle regulation. Moreover, Flp1 contributes to checkpoint activation during replication stress. Here, we show that Flp1 has a role in fine-tuning the cellular oxidative stress response. Phosphorylation-dependent nucleolar release of Flp1 in response to oxidative stress conditions plays a role in the cellular transcriptional response. Thus, Flp1 ablation increases the transcriptional response to oxidative stress, in both intensity and duration, upregulating both Atf1/Pcr1- and Pap1-dependent stress induced genes. Remarkably, we found that Flp1 interacts with the Atf1/Pcr1 complex with Pcr1 acting as a direct substrate. Our results provide evidence that Flp1 modulates the oxidative stress response by limiting the Atf1/Pcr1-mediated transcription.

Transcriptional activation of the p53 target genes plays a critical role in the cellular response to DNA damage, hypoxia, cellular stress and other signals regulating the cell cycle and apoptosis. The discovery of new p53 target genes continues to reveal novel mechanisms of action of this multifaceted protein. We used cDNA arrays to search for p53-regulated genes in prostate cancer cells. In this report, we describe robust induction of heat shock protein 27 (hsp27) in prostate cancer cells (DU145, LNCaP, PC3) following wild-type p53 expression from an adenoviral p53 expression vector (AdWTp53). A mutant p53 (R175H)-containing adenoviral expression vector did not induce hsp27. hsp27 expression was not altered in prostate cancer cells following expression of cyclin-dependent kinase inhibitors: p21(waf1/cip1) and p27(kip1) from adenoviral expression vectors. Treatment of cells with staurosporine, an apoptosis-inducing agent, did no affect hsp27 expression. These observations provide evidence that induction of hsp27 expression was wild-type p53-specific and was not due to non-specific effects of cell growth arrest and/or apoptosis. Previous studies and the experiment reported here show induction of hsp27 expression in response to androgen ablation, a physiological state that induces apoptosis in prostatic epithelial cells. The nature of p53 and hsp27 interactions in the regulation of apoptosis and/or cell growth needs to be further defined.

Understanding the chronic thermal acclimation capacity of chum salmon (Oncorhynchus keta) is essential for predicting species resilience and developing mitigation strategies under ocean warming. We investigated the upper limit of chronic thermal acclimation and its underlying molecular mechanisms in chum salmon smolts exposed to four constant temperatures (10, 14, 18, and 22 °C) for 6 weeks. Transcriptional responses of genes related to cellular stress protection, endocrine feedback regulation, antioxidant defense, metabolic regulation (AMPKα and mTOR), and protein degradation were quantified in the liver, skeletal muscle, and brain. Chronic exposure to elevated temperature elicited tissue-specific molecular responses, with the most pronounced effects observed at 22 °C. At this temperature, all tissues showed marked induction of heat shock proteins and ubiquitin, accompanied by suppression of antioxidant defenses, glucocorticoid receptor signaling, and AMPKα–mTOR-mediated metabolic regulation, particularly in the liver and muscle. These responses were consistent with previously reported impairments in growth performance, lipid reserves, and hematological indices from the same growth trial. In contrast, smolts maintained at 18 °C exhibited molecular signatures indicative of effective physiological compensation without severe cellular stress. Collectively, these results indicate that chum salmon smolts can acclimate to chronic warming up to 18 °C, whereas exposure to 22 °C exceeds their acclimation capacity and induces a tertiary stress response.