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1.
Proc Natl Acad Sci U S A ; 119(30): e2122140119, 2022 07 26.
Artículo en Inglés | MEDLINE | ID: mdl-35867837

RESUMEN

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.


Asunto(s)
Hipertermia Maligna , Músculo Esquelético , Miopatía del Núcleo Central , Canal Liberador de Calcio Receptor de Rianodina , Animales , Calcio/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Hipertermia Maligna/genética , Músculo Esquelético/metabolismo , Mutación , Miopatía del Núcleo Central/genética , Conejos , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genética
2.
IUBMB Life ; 75(11): 926-940, 2023 11.
Artículo en Inglés | MEDLINE | ID: mdl-37427864

RESUMEN

Frequent premature ventricular contractions (PVCs) promoted eccentric cardiac hypertrophy and reduced ejection fraction (EF) in a large animal model of PVC-induced cardiomyopathy (PVC-CM), but the molecular mechanisms and markers of this hypertrophic remodeling remain unexplored. Healthy mongrel canines were implanted with pacemakers to deliver bigeminal PVCs (50% burden with 200-220 ms coupling interval). After 12 weeks, left ventricular (LV) free wall samples were studied from PVC-CM and Sham groups. In addition to reduced LV ejection fraction (LVEF), the PVC-CM group showed larger cardiac myocytes without evident ultrastructural alterations compared to the Sham group. Biochemical markers of pathological hypertrophy, such as store-operated Ca2+ entry, calcineurin/NFAT pathway, ß-myosin heavy chain, and skeletal type α-actin were unaltered in the PVC-CM group. In contrast, pro-hypertrophic and antiapoptotic pathways including ERK1/2 and AKT/mTOR were activated and/or overexpressed in the PVC-CM group, which appeared counterbalanced by an overexpression of protein phosphatase 1 and a borderline elevation of the anti-hypertrophic factor atrial natriuretic peptide. Moreover, the potent angiogenic and pro-hypertrophic factor VEGF-A and its receptor VEGFR2 were significantly elevated in the PVC-CM group. In conclusion, a molecular program is in place to keep this structural remodeling associated with frequent PVCs as an adaptive pathological hypertrophy.


Asunto(s)
Cardiomiopatías , Complejos Prematuros Ventriculares , Animales , Perros , Complejos Prematuros Ventriculares/complicaciones , Remodelación Ventricular , Modelos Animales de Enfermedad , Hipertrofia/complicaciones
3.
Mol Cell Biochem ; 478(7): 1447-1456, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-36350464

RESUMEN

Premature ventricular contractions (PVCs) are the most frequent ventricular arrhythmias in the overall population. PVCs are known to acutely enhance contractility by the post-extrasystolic potentiation phenomenon, but over time persistent PVCs promote PVC-induced cardiomyopathy (PVC-CM), characterized by a reduction of the left ventricular (LV) ejection fraction. Ca2+ cycling in myocytes commands muscle contraction and in this process, SERCA2 leads the Ca2+ reuptake into the sarcoplasmic reticulum (SR) shaping cytosolic Ca2+ signal decay and muscle relaxation. Altered Ca2+ reuptake can contribute to the contractile dysfunction observed in PVC-CM. To better understand Ca2+ handling using our PVC-CM model (canines with 50% PVC burden for 12 weeks), SR-Ca2+ reuptake was investigated by measuring Ca2+ dynamics and analyzing protein expression. Kinetic analysis of Ca2+ reuptake in electrically paced myocytes showed a ~ 21 ms delay in PVC-CM compared to Sham in intact isolated myocytes, along with a ~ 13% reduction in SERCA2 activity assessed in permeabilized myocytes. Although these trends were not statistically significant between groups using hierarchical statistics, relaxation of myocytes following contraction was significantly slower in PVC-CM vs Sham myocytes. Western blot analyses indicate a 22% reduction in SERCA2 expression, a 23% increase in phospholamban (PLN) expression, and a 50% reduction in PLN phosphorylation in PVC-CM samples vs Sham. Computational analysis simulating a 20% decrease in SR-Ca2+ reuptake resulted in a ~ 22 ms delay in Ca2+ signal decay, consistent with the experimental result described above. In conclusion, SERCA2 and PLB alterations described above have a modest contribution to functional adaptations observed in PVC-CM.


Asunto(s)
Cardiomiopatías , Complejos Prematuros Ventriculares , Animales , Perros , Complejos Prematuros Ventriculares/metabolismo , Retículo Sarcoplasmático/metabolismo , Cinética , Cardiomiopatías/metabolismo , Células Musculares , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Calcio/metabolismo , Miocitos Cardíacos/metabolismo
4.
J Struct Biol ; 205(2): 180-188, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30641143

RESUMEN

Ryanodine receptors (RyRs) are large conductance intracellular channels controlling intracellular calcium homeostasis in myocytes, neurons, and other cell types. Loss of RyR's constitutive cytoplasmic partner FKBP results in channel sensitization, dominant subconductance states, and increased cytoplasmic Ca2+. FKBP12 binds to RyR1's cytoplasmic assembly 130 Šaway from the ion gate at four equivalent sites in the RyR1 tetramer. To understand how FKBP12 binding alters RyR1's channel properties, we studied the 3D structure of RyR1 alone in the closed conformation in the context of the open and closed conformations of FKBP12-bound RyR1. We analyzed the metrics of conformational changes of existing structures, the structure of the ion gate, and carried out multivariate statistical analysis of thousands of individual cryoEM RyR1 particles. We find that under closed state conditions, in the presence of FKBP12, the cytoplasmic domain of RyR1 adopts an upward conformation, whereas absence of FKBP12 results in a relaxed conformation, while the ion gate remains closed. The relaxed conformation is intermediate between the RyR1-FKBP12 complex closed (upward) and open (downward) conformations. The closed-relaxed conformation of RyR1 appears to be consistent with a lower energy barrier separating the closed and open states of RyR1-FKBP12, and suggests that FKBP12 plays an important role by restricting conformations within RyR1's conformational landscape.


Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteína 1A de Unión a Tacrolimus/química , Proteína 1A de Unión a Tacrolimus/metabolismo , Animales , Microscopía por Crioelectrón , Humanos , Unión Proteica , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Proteína 1A de Unión a Tacrolimus/genética
6.
Biophys J ; 110(12): 2651-2662, 2016 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-27332123

RESUMEN

In heart, type-2 ryanodine receptor (RyR2) forms discrete supramolecular clusters in the sarcoplasmic reticulum known as calcium release units (CRUs), which are responsible for most of the Ca(2+) released for muscle contraction. To learn about the substructure of the CRU, we sought to determine whether RyR2s have the ability to self-associate in the absence of other factors and if so, whether they do it in a specific manner. Purified RyR2 was negatively stained and imaged on the transmission electron microscope, and RyR2 particles closely associated were further analyzed using bias-free multivariate statistical analysis and classification. The resulting two-dimensional averages show that RyR2s can interact in two rigid, reproducible configurations: "adjoining", with two RyR2s alongside each other, and "oblique", with two partially overlapped RyR2s forming an angle of 12°. The two configurations are nearly identical under two extreme physiological Ca(2+) concentrations. Pseudo-atomic models for these two interactions indicate that the adjoining interaction involves contacts between the P1, SPRY1 and the helical domains. The oblique interaction is mediated by extensive contacts between the SPRY1 domains (domains 9) and P1 domains (domains 10) of both RyR2s and not through domain 6 as previously thought; in addition its asymmetric interface imposes steric constrains that inhibit the growth of RyR2 as a checkerboard, which is the configuration usually assumed, and generates new configurations, i.e., "branched" and "interlocked". This first, to our knowledge, structural detailed analysis of the inter-RyR2 interactions helps to understand important morphological and functional aspects of the CRU in the context of cardiac EC coupling.


Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Animales , Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Ventrículos Cardíacos/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica de Transmisión , Modelos Moleculares , Análisis Multivariante , Dominios Proteicos , Multimerización de Proteína , Canal Liberador de Calcio Receptor de Rianodina/aislamiento & purificación , Sus scrofa , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo
7.
J Biol Chem ; 288(9): 6154-65, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23319589

RESUMEN

Ryanodine receptor type 1 (RyR1) releases Ca(2+) from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca(2+). However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912-Glu-4948) and a linker helix (Val-4830-Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel.


Asunto(s)
Activación del Canal Iónico , Modelos Moleculares , Músculo Esquelético/química , Canal Liberador de Calcio Receptor de Rianodina/química , Sustitución de Aminoácidos , Animales , Células HEK293 , Humanos , Músculo Esquelético/metabolismo , Mutación Missense , Estructura Terciaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
8.
J Phys Chem B ; 128(19): 4670-4684, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38717304

RESUMEN

Ryanodine receptor type 1 (RyR1) is a Ca2+-release channel central to skeletal muscle excitation-contraction (EC) coupling. RyR1's cryo-EM structures reveal a zinc-finger motif positioned within the cytoplasmic C-terminal domain (CTD). Yet, owing to limitations in cryo-EM resolution, RyR1 structures lack precision in detailing the metal coordination structure, prompting the need for an accurate model. In this study, we employed molecular dynamics (MD) simulations and the density functional theory (DFT) method to refine the binding characteristics of Zn2+ in the zinc-finger site of the RyR1 channel. Our findings also highlight substantial conformational changes in simulations conducted in the absence of Zn2+. Notably, we observed a loss of contact at the interface between protein domains proximal to the zinc-finger site, indicating a crucial role of Zn2+ in maintaining structural integrity and interdomain interactions within RyR1. Furthermore, this study provides valuable insights into the modulation of ATP, Ca2+, and caffeine binding, shedding light on the intricate relationship between Zn2+ coordination and the dynamic behavior of RyR1. Our integrative approach combining MD simulations and DFT calculations enhances our understanding of the molecular mechanisms governing ligand binding in RyR1.


Asunto(s)
Simulación de Dinámica Molecular , Canal Liberador de Calcio Receptor de Rianodina , Zinc , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Zinc/química , Zinc/metabolismo , Ligandos , Calcio/química , Calcio/metabolismo , Teoría Funcional de la Densidad , Sitios de Unión , Unión Proteica , Dedos de Zinc , Cafeína/química , Cafeína/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Humanos
9.
J Gen Physiol ; 156(9)2024 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-38980209

RESUMEN

Skeletal muscle, the major processor of dietary glucose, stores it in myriad glycogen granules. Their numbers vary with cellular location and physiological and pathophysiological states. AI models were developed to derive granular glycogen content from electron-microscopic images of human muscle. Two UNet-type semantic segmentation models were built: "Locations" classified pixels as belonging to different regions in the cell; "Granules" identified pixels within granules. From their joint output, a pixel fraction pf was calculated for images from patients positive (MHS) or negative (MHN) to a test for malignant hyperthermia susceptibility. pf was used to derive vf, the volume fraction occupied by granules. The relationship vf (pf) was derived from a simulation of volumes ("baskets") containing virtual granules at realistic concentrations. The simulated granules had diameters matching the real ones, which were measured by adapting a utility devised for calcium sparks. Applying this relationship to the pf measured in images, vf was calculated for every region and patient, and from them a glycogen concentration. The intermyofibrillar spaces and the sarcomeric I band had the highest granular content. The measured glycogen concentration was low enough to allow for a substantial presence of non-granular glycogen. The MHS samples had an approximately threefold lower concentration (significant in a hierarchical test), consistent with earlier evidence of diminished glucose processing in MHS. The AI models and the approach to infer three-dimensional magnitudes from two-dimensional images should be adaptable to other tasks on a variety of images from patients and animal models and different disease conditions.


Asunto(s)
Glucógeno , Músculo Esquelético , Humanos , Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/diagnóstico por imagen , Inteligencia Artificial , Microscopía Electrónica/métodos
10.
Nat Commun ; 15(1): 5120, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38879623

RESUMEN

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.


Asunto(s)
Calcio , Calmodulina , Transferencia Resonante de Energía de Fluorescencia , Músculo Esquelético , Miocardio , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Calmodulina/metabolismo , Calmodulina/química , Calcio/metabolismo , Miocardio/metabolismo , Cinética , Animales , Músculo Esquelético/metabolismo , Humanos , Conformación Proteica , Unión Proteica , Retículo Sarcoplasmático/metabolismo
11.
Nat Commun ; 15(1): 4115, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38750013

RESUMEN

RyR1 is an intracellular Ca2+ channel important in excitable cells such as neurons and muscle fibers. Ca2+ activates it at low concentrations and inhibits it at high concentrations. Mg2+ is the main physiological RyR1 inhibitor, an effect that is overridden upon activation. Despite the significance of Mg2+-mediated inhibition, the molecular-level mechanisms remain unclear. In this work we determined two cryo-EM structures of RyR1 with Mg2+ up to 2.8 Å resolution, identifying multiple Mg2+ binding sites. Mg2+ inhibits at the known Ca2+ activating site and we propose that the EF hand domain is an inhibitory divalent cation sensor. Both divalent cations bind to ATP within a crevice, contributing to the precise transmission of allosteric changes within the enormous channel protein. Notably, Mg2+ inhibits RyR1 by interacting with the gating helices as validated by molecular dynamics. This structural insight enhances our understanding of how Mg2+ inhibition is overcome during excitation.


Asunto(s)
Calcio , Microscopía por Crioelectrón , Magnesio , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Magnesio/metabolismo , Calcio/metabolismo , Sitios de Unión , Animales , Simulación de Dinámica Molecular , Adenosina Trifosfato/metabolismo , Humanos , Conejos
12.
J Biol Chem ; 287(52): 43853-61, 2012 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-23118233

RESUMEN

The L-type Ca(2+) channel (dihydropyridine receptor (DHPR) in skeletal muscle acts as the voltage sensor for excitation-contraction coupling. To better resolve the spatial organization of the DHPR subunits (α(1s) or Ca(V)1.1, α(2), ß(1a), δ1, and γ), we created transgenic mice expressing a recombinant ß(1a) subunit with YFP and a biotin acceptor domain attached to its N- and C- termini, respectively. DHPR complexes were purified from skeletal muscle, negatively stained, imaged by electron microscopy, and subjected to single-particle image analysis. The resulting 19.1-Å resolution, three-dimensional reconstruction shows a main body of 17 × 11 × 8 nm with five corners along its perimeter. Two protrusions emerge from either face of the main body: the larger one attributed to the α(2)-δ1 subunit that forms a flexible hook-shaped feature and a smaller protrusion on the opposite side that corresponds to the II-III loop of Ca(V)1.1 as revealed by antibody labeling. Novel features discernible in the electron density accommodate the atomic coordinates of a voltage-gated sodium channel and of the ß subunit in a single docking possibility that defines the α1-ß interaction. The ß subunit appears more closely associated to the membrane than expected, which may better account for both its role in localizing the α(1s) subunit to the membrane and its suggested role in excitation-contraction coupling.


Asunto(s)
Canales de Calcio Tipo L/ultraestructura , Simulación del Acoplamiento Molecular , Proteínas Musculares/ultraestructura , Músculo Esquelético/ultraestructura , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Humanos , Ratones , Ratones Transgénicos , Microscopía Electrónica , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína
13.
Curr Opin Pharmacol ; 68: 102327, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36516687

RESUMEN

Mutations in RyR alter the cell's Ca2+ homeostasis and can cause serious health problems for which few effective therapies are available. Until recently, there was little structural context for the hundreds of mutations linked to muscular disorders reported for this large channel. Growing knowledge of the three-dimensional structure of RyR starts to illustrate the fine control of Ca2+ release. Current efforts directed towards understanding how disease mutations impinge in such processes will be crucial for future design of novel therapies. In this review article we discuss the up-to-date information about mutations according to their role in the 3D structure, and classified them to provide context from a structural perspective.


Asunto(s)
Señalización del Calcio , Canal Liberador de Calcio Receptor de Rianodina , Humanos , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Mutación , Miocardio/metabolismo , Homeostasis , Calcio/metabolismo , Músculo Esquelético/metabolismo
14.
PLoS Biol ; 7(4): e85, 2009 Apr 14.
Artículo en Inglés | MEDLINE | ID: mdl-19402748

RESUMEN

Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices"). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.


Asunto(s)
Activación del Canal Iónico , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Secuencia de Aminoácidos , Animales , Quelantes/farmacología , Microscopía por Crioelectrón , Ácido Egtácico/farmacología , Contaminantes Ambientales/farmacología , Canal de Potasio Kv.1.2/química , Modelos Moleculares , Datos de Secuencia Molecular , Bifenilos Policlorados/farmacología , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Proteína 1A de Unión a Tacrolimus/metabolismo
15.
Elife ; 112022 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-35257661

RESUMEN

Activation of the intracellular Ca2+ channel ryanodine receptor (RyR) triggers a cytosolic Ca2+ surge, while elevated cytosolic Ca2+ inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca2+ conditions revealed an open and a closed-inactivated conformation. Ca2+ binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca2+-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca2+ activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca2+ activation prerequisite for Ca2+ inactivation and provides for a seamless transition from inactivated to closed conformations.


Asunto(s)
Calcio , Canal Liberador de Calcio Receptor de Rianodina , Animales , Calcio/metabolismo , Microscopía por Crioelectrón , Citosol/metabolismo , Motivos EF Hand , Mamíferos/metabolismo , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
16.
mBio ; 13(6): e0227022, 2022 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-36326250

RESUMEN

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.


Asunto(s)
Escherichia coli Enteropatógena , Humanos , Escherichia coli Enteropatógena/metabolismo , Adenosina Trifosfatasas/metabolismo , Microscopía por Crioelectrón , Adenilil Imidodifosfato/análisis , Adenilil Imidodifosfato/metabolismo , Fimbrias Bacterianas/metabolismo , Proteínas Fimbrias/metabolismo
17.
J Biol Chem ; 285(25): 19219-26, 2010 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-20404344

RESUMEN

The 12-kDa FK506-binding proteins (FKBP12 and FKBP12.6) are regulatory subunits of ryanodine receptor (RyR) Ca(2+) release channels. To investigate the structural basis of FKBP interactions with the RyR1 and RyR2 isoforms, we used site-directed fluorescent labeling of FKBP12.6, ligand binding measurements, and fluorescence resonance energy transfer (FRET). Single-cysteine substitutions were introduced at five positions distributed over the surface of FKBP12.6. Fluorescent labeling at position 14, 32, 49, or 85 did not affect high affinity binding to the RyR1. By comparison, fluorescent labeling at position 41 reduced the affinity of FKBP12.6 binding by 10-fold. Each of the five fluorescent FKBPs retained the ability to inhibit [(3)H]ryanodine binding to the RyR1, although the maximal extent of inhibition was reduced by half when the label was attached at position 32. The orientation of FKBP12.6 bound to the RyR1 and RyR2 was examined by measuring FRET from the different labeling positions on FKBP12.6 to an acceptor attached within the RyR calmodulin subunit. FRET was dependent on the position of fluorophore attachment on FKBP12.6; however, for any given position, the distance separating donors and acceptors bound to RyR1 versus RyR2 did not differ significantly. Our results show that FKBP12.6 binds to RyR1 and RyR2 in the same orientation and suggest new insights into the discrete structural domains responsible for channel binding and inhibition. FRET mapping of RyR-bound FKBP12.6 is consistent with the predictions of a previous cryoelectron microscopy study and strongly supports the proposed structural model.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Canal Liberador de Calcio Receptor de Rianodina/química , Proteínas de Unión a Tacrolimus/química , Animales , Calmodulina/química , Humanos , Inmunofilinas/química , Músculo Esquelético/metabolismo , Mutación , Miocardio/patología , Unión Proteica , Estructura Terciaria de Proteína , Rianodina/química , Retículo Sarcoplasmático/metabolismo , Porcinos
18.
Bio Protoc ; 11(15): e4112, 2021 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-34458406

RESUMEN

High quantities of purified ryanodine receptor (RyR), a large (2.26 MDa) intracellular homotetrameric membrane protein, can be obtained from heterologous expression in HEK293 cells and used for structure determination by cryo-EM. The advantage of using recombinant protein is that the variability due to post-translational modifications can be minimized, to which the high resolution of up to 2.4 Å achieved for RyR2 can be attributed ( Iyer et al., 2020 ). In addition, recombinant protein expression enables the study of mutations that are deleterious when expressed homozygously in animals. Protein purification was achieved using two strategies, sucrose density gradient and affinity chromatography, which have previously been used for purification of RyR from tissue. The sucrose gradient method was developed from ( Lee et al., 1994 ) and later adapted for cryo-EM ( Samsó et al., 2005 ). The affinity chromatography method takes advantage of the high affinity of RyR for its ligand FKBP12/12.6, by using a construct between FKBP and streptavidin binding protein (SBP) ( Cabra et al., 2016 ). While the sucrose gradient method can yield a higher protein concentration (≥ 2 mg/ml), the affinity purification method is faster. Both methods are suitable and applicable to the purification of recombinant proteins and were successfully used in the first 3D near-atomic reconstructions of RyRs purified from cells expressing disease mutants ( Iyer et al., 2020 ). This purification protocol is also suitable for functional studies, such as single-channel analysis, that require pure RyR protein.

19.
Nat Struct Mol Biol ; 12(6): 539-44, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15908964

RESUMEN

RyR1 is an intracellular calcium channel with a central role in muscle contraction. We obtained a three-dimensional reconstruction of the RyR1 in the closed state at a nominal resolution of approximately 10 A using cryo-EM. The cytoplasmic assembly consists of a series of interconnected tubular structures that merge into four columns that extend into the transmembrane assembly. The transmembrane assembly, which has at least six transmembrane alpha-helices per monomer, has four tilted rods that can be fitted with the inner helices of a closed K(+) channel atomic structure. The rods splay out at the lumenal side and converge into a dense ring at the cytoplasmic side. Another set of four rods emerges from this ring and shapes the inner part of the four columns. The resulting constricted axial structure provides direct continuity between cytoplasmic and transmembrane assemblies, and a possible mechanism for control of channel gating through conformational changes in the cytoplasmic assembly.


Asunto(s)
Membrana Celular/ultraestructura , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Animales , Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador , Modelos Moleculares , Músculo Esquelético/ultraestructura , Conformación Proteica , Conejos , Canal Liberador de Calcio Receptor de Rianodina/química , Sensibilidad y Especificidad
20.
Sci Adv ; 6(31): eabb2964, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32832689

RESUMEN

Mutations in ryanodine receptors (RyRs), intracellular Ca2+ channels, are associated with deadly disorders. Despite abundant functional studies, the molecular mechanism of RyR malfunction remains elusive. We studied two single-point mutations at an equivalent site in the skeletal (RyR1 R164C) and cardiac (RyR2 R176Q) isoforms using ryanodine binding, Ca2+ imaging, and cryo-electron microscopy (cryo-EM) of the full-length protein. Loss of the positive charge had greater effect on the skeletal isoform, mediated via distortion of a salt bridge network, a molecular latch inducing rotation of a cytoplasmic domain, and partial progression to open-state traits of the large cytoplasmic assembly accompanied by alteration of the Ca2+ binding site, which concur with the major "hyperactive" feature of the mutated channel. Our cryo-EM studies demonstrated the allosteric effect of a mutation situated ~85 Å away from the pore and identified an isoform-specific structural effect.


Asunto(s)
Mutación con Ganancia de Función , Canal Liberador de Calcio Receptor de Rianodina , Microscopía por Crioelectrón , Músculo Esquelético/metabolismo , Mutación , Dominios Proteicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo
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