Structural and Functional Characterization of Phospholipase C β3

SAXS Ensemble Refinement of ESCRT-III CHMP3 Conformational Transitions

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

Striatin (Strn) is an important scaffolding protein linked to various cardiovascular diseases, including arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy, and hypertrophic cardiomyopathy, as demonstrated in preclinical1-3 studies. The use of cardiomyocytes derived from embryonic stem cells and induced pluripotent stem cells has become increasingly valuable for studying the molecular pathomechanisms underlying these cardiomyopathies. These models closely resemble human cardiomyocytes and possess the ability to differentiate into various cell4-9 types, providing a robust platform for preclinical studies and drug screening. Despite their inherent limitations, such as the lack of hormonal or neural support, these models have successfully elucidated several channelopathies and cardiomyopathies.4-11 Strn is characterized by four distinct protein–protein interaction domains, yet its functional role in cardiomyocytes remains inadequately explored. Notably, the specific impact of Strn on ion channel currents within cardiomyocytes has not been investigated until now. The study by Benzoni et al.12 provides the first evidence of Strn's functional significance through a knockout model in cardiomyocytes derived from embryoid bodies. The authors observe critical dysregulation in contraction dynamics and intracellular calcium handling, alongside a higher beating rate and alterations in action potential characteristics. Their findings reveal increased densities of both transient and late sodium channel currents in the Strn knockout model. Furthermore, the authors analyze INCX activity, noting a reduction in the knockout model compared to wild-type cardiomyocytes, which suggests a potential interplay between sodium channel currents and calcium homeostasis. Benzoni et al. propose that these observed alterations stem from a dysregulated cytoskeletal network and ion channel function due to the absence of Strn. Intriguingly, the study also explores the effects of taxol, a significant inhibitor of the late sodium current, which reverses the observed changes in the knockout model. This finding underscores the multifaceted role of Strn in not only mediating functional changes in heart muscle but also modulating broader aspects of cellular physiology. Previous studies have indicated that cardiac Strn interacts with caveolin-3 and calmodulin in a calcium-sensitive manner, which regulates the spontaneous contraction13 rate of cardiomyocytes (Figure 1). Additionally, genome-wide association studies have linked the localization of Strn to changes in QRS duration, suggesting its potential involvement in heart rhythm disorders and sudden cardiac death.14 However, comprehensive studies involving larger patient cohorts are necessary to fully elucidate the role of Strn in these pathologies. Overall, the findings presented by Benzoni et al. offer the first mechanistic evidence that the absence of Strn significantly alters both the electrical and mechanical properties of cardiomyocytes. To further understand the implications of Strn in clinical settings, large cohort studies of patients with cardiomyopathies are essential. By leveraging bioinformatics, human genetic data, and animal or in vitro models, a predictive model for Strn's role in cardiovascular disease could be developed. These insights could pave the way for establishing novel therapeutic targets for affected patients. Future research should also explore the specific signaling pathways that Strn influences within cardiomyocytes, particularly focusing on its interactions with other proteins involved in calcium signaling and contractile function. Investigating the relationship between Strn and other known modulators of cardiac function could provide a more comprehensive understanding of its role in cardiovascular health and disease. Furthermore, the potential for Strn to serve as a biomarker for cardiovascular diseases should be examined, as its expression levels may correlate with disease severity or patient outcomes. Identifying such biomarkers could facilitate early diagnosis and personalized treatment strategies for patients suffering from heart diseases. In hypertrophic cardiomyopathy, for instance, mavacamten, a myosin inhibitor, is currently the only drug shown to decrease pressure in the left ventricular outflow tract. However, there remains a significant need for additional therapeutic options. Exploring the role of each protein domain of Strn in preclinical settings could yield critical evidence for developing innovative treatment strategies. Various approaches should be employed to delineate the specific role of each domain, identifying which domain exerts the most significant influence on Strn's overall function. This knowledge could be pivotal in devising targeted therapies that address the underlying mechanisms of cardiovascular diseases, ultimately improving patient outcomes. Ibrahim El-Battrawy: Writing – original draft. Nazha Hamdani: Writing – review and editing. Ibrahim Akin: Writing – review and editing. The authors have nothing to report. The research was funded by EU’s Horizon 2020 research and innovation program under grant agreement No. 739593 to N.H.; DFG (Deutsche Forschungsgemeinschaft) HA 7512/2-4 and HA 7512/2-1 to N.H.; a grant from the Innovation Forum program of the Medical Faculty, RUB to I. EL-B. and N.H No. IF-023-22 and No. IF-034-22; and the Hector-Stiftung (No.; MED1814, M2401) to N.H, I. EL-B, and I.A and the German Heart foundation (F/17/23) to I. El-B. and funding from Else-Kröner-Fresenius foundation to I. El-B. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

<b>Abstract ID 23793</b> <b>Poster Board 164</b> G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signaling events through activation of the heterotrimeric G protein subunits, Gα and Gβγ. G proteins in turn activate effector enzymes, such as phospholipase C β (PLCβ), to produce second messengers. The four human PLCβ isoforms (PLCb1-4) are all potently activated by direct binding of Gα_q, but only PLCβ1-3 are also directly activated by Gβγ. Dysregulation of the G protein-phospholipase C signaling axis has been implicated in numerous pathologies and disease states. Direct inhibition of these pathways has shown therapeutic potential in models of inflammation, cardiac hypertrophy, opioid analgesia, and cancer. Much is known about how Gα_qbinds to and activates PLCβ, but despite decades of research, the molecular details of Gβγ-stimulated activation have been elusive. Using a protein engineering strategy and single particle cryo-electron microscopy, we have now determined high-resolution structures of functional Gβγ–PLCβ3 complexes. The structures reveal that the Gβγ interacts with PLCβ3 through multivalent interactions with the PH domain, EF hands and the C2 domains of PLCβ. The structures differ in the engagement of the Gβγ subunit with the PH-EF hand interface. Intriguingly, one conformation of the Gβγ–PLCβ complex is compatible with simultaneous binding of Gα_i, which has not been observed for any other Gβγ–effector enzyme complex. Cellular activity assays suggest that this conformation is less sensitive to Gβγ-dependent activation, and, in assays with purified components, that crosslinked Gβγ-PLCβ complexes still sensitive to Gα_i inhibition. Thus, Gβγ or the Gα_iβγ complex may also function as a scaffold for PLCβ, in which a pre-activated complex is maintained at the membrane. Current efforts in the lab include investigating the scaffolding function of these complexes and PLCβ insensitivity to Gβγ activation.

Editorial (Hot Topic: New Insight into the RAMP-based Family B GPCRs and their Peptide Ligands)

The 826 G protein-coupled receptors (GPCRs) in the human proteome regulate key physiological processes and thus have long been attractive drug targets. With the crystal structures of more than 50 different human GPCRs determined over the past decade, an initial platform for structure-based rational design has been established for drugs that target GPCRs, which is currently being augmented with cryo-electron microscopy (cryo-EM) structures of higher-order GPCR complexes. Nuclear magnetic resonance (NMR) spectroscopy in solution is one of the key approaches for expanding this platform with dynamic features, which can be accessed at physiological temperature and with minimal modification of the wild-type GPCR covalent structures. Here, we review strategies for the use of advanced biochemistry and NMR techniques with GPCRs, survey projects in which crystal or cryo-EM structures have been complemented with NMR investigations and discuss the impact of this integrative approach on GPCR biology and drug discovery.

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

Obesity has been described as the biggest health threat facing the Western world, and this statement will probably need to be revised shortly to encompass the entire world. The social and economic impact of this pandemic is enormous. Currently, a third of all Americans are obese, and this may reach

Introduction Cardiovascular disease (CVD) is the leading cause of morbidity globally, with chronic inflammation as a key modifiable risk factor. Toll-like receptors (TLRs), pivotal components of the innate immune system, including TLR-3, −7, −8, and −9 within endosomes, trigger intracellular cascades, leading to inflammatory cytokine production by various cell types, contributing to systemic inflammation and atherosclerosis. Recent research highlights the role of endosomal TLRs in recognizing self-derived nucleic acids during sterile inflammation, implicated in autoimmune conditions like myocarditis. Areas covered This review explores the impact of endosomal TLRs on viral infections, autoimmunity, and inflammatory responses, shedding light on their intricate involvement in cardiovascular health and disease by examining literature on TLR-mediated mechanisms and their roles in CVD pathophysiology. Expert opinion Removal of endosomal TLRs mitigates myocardial damage and immune reactions, applicable in myocardial injury. Targeting TLRs with agonists enhances innate immunity against fatal viruses, lowering viral loads and mortality. Prophylactic TLR agonist administration upregulates TLRs, protecting against fatal viruses and improving survival. TLRs play a complex role in CVDs like atherosclerosis and myocarditis, with therapeutic potential in modulating TLR reactions for cardiovascular health.

Regular physical activity is widely accepted as playing a crucial role in cardiovascular disease (CVD) prevention. However, despite extensive research in this field there are crucial gaps in our knowledge. This paper discusses future research directions including population subgroup-specific exercise doses, research in under represented populations, the role of cardiorespiratory fitness in predicting CVD risk, and novel biological mechanisms that might mediate the inverse association between physical activity and CVD risk.

Development of imaging scaffolds for cryo-electron microscopy

Histotoxic clostridia produce collagenases responsible for extensive tissue destruction in gas gangrene. The C-terminal collagen-binding domain (CBD) of these enzymes is the minimal segment required to bind to collagen fibril. Collagen binding efficiency of CBD is more pronounced in the presence of Ca(2+). We have shown that CBD can be functional to anchor growth factors in local tissue. A (1)H-(15)N HSQC NMR titration study with three different tropocollagen analogues ((POG)(10))(3), ((GPOG)(7)PRG)(3), and (GPRG(POG)(7)C-carbamidomethyl)(3), mapped a saddle-like binding cleft on CBD. NMR titrations with three nitroxide spin-labeled analogues of collagenous peptide, (PROXYL-G(POG)(7)PRG)(3), (PROXYL-G(POG)(7))(3), and (GPRG(POG)(7)C-PROXYL)(3) (where PROXYL represents 2,2,5,5-tetramethyl-l-pyrrolidinyloxy), unambiguously demonstrated unidirectional binding of CBD to the tropocollagen analogues. Small angle x-ray scattering data revealed that CBD binds closer to a terminus for each of the five different tropocollagen analogues, which in conjunction with NMR titration studies, implies a binding mode where CBD binds to the C terminus of the triple helix.

Single Particle Kinetic and Electron Cryo-Microscopy Studies of Flavivirus Membrane Fusion

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.