scholarly journals Structural mechanism of two gain-of-function cardiac and skeletal RyR mutations at an equivalent site by cryo-EM

2020 ◽  
Vol 6 (31) ◽  
pp. eabb2964 ◽  
Author(s):  
Kavita A. Iyer ◽  
Yifan Hu ◽  
Ashok R. Nayak ◽  
Nagomi Kurebayashi ◽  
Takashi Murayama ◽  
...  
Keyword(s):  
Single Point ◽  
Ryanodine Receptors ◽  
Point Mutations ◽  
Salt Bridge ◽  
Structural Effect ◽  
Protein Loss ◽  
Structural Mechanism ◽  
Functional Studies ◽  
Equivalent Site ◽  
Intracellular Ca

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.

scholarly journals Cryo-EM reveals local and global structural rearrangements in RYR mutants

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Kavita A. Iyer ◽  
Yifan Hu ◽  
Thomas Klose ◽  
Takashi Murayama ◽  
Montserrat Samsó
Keyword(s):  
Long Range ◽  
Conformational Changes ◽  
Large Scale ◽  
Single Point ◽  
Ryanodine Receptors ◽  
Critical Role ◽  
Point Mutations ◽  
Polymorphic Ventricular Tachycardia ◽  
Functional Studies ◽  
Single Point Mutations

Single-point mutations in ryanodine receptors (RYRs), large intracellular Ca2+ channels that play a critical role in EC coupling, are linked to debilitating and lethal disorders such as central core disease, malignant hyperthermia (for the skeletal isoform, RYR1), catecholaminergic polymorphic ventricular tachycardia, and ARVD2 (for the cardiac isoform, RYR2). Mutant RYRs result in elevated [Ca2+]cyto due to steady leak from the sarcoplasmic reticulum. To explore the nature of long-range allosteric mechanisms of malfunction, we determined the structure of two N-terminal domain mutants of RYR1, situated far away from the pore. Cryo-electron microscopy of the N-terminal subdomain A (NTDA) and subdomain C (NTDC) full-length mutants, RYR1 R163C (determined to 3.5 Å resolution), and RYR1 Y522S (determined to 4.0 Å resolution), respectively, reveal large-scale conformational changes in the cytoplasmic assembly under closed-state conditions (i.e., absence of activating Ca2+). The multidomain changes suggest that the mutations induce a preactivated state of the channel in R164C by altering the NTDA+/CD interface, and in Y522S by rearrangement of the α-helical bundle in NTDC. However, the extent of preactivation is considerably higher in Y522S as compared with R163C, which agrees with the increased severity of the Y522S mutation as established by various functional studies. The Y522S mutation represents loss of a spacer residue that is crucial for maintaining optimal orientation of α helices in NTDC, alteration of which has long-range effects felt as far away as ∼100 Å. Additionally, the structure of the Y522S mutant channel under open-state conditions also differs from RYR1 WT open channels. Our developing work with RYR mutants exhibits the diverse mechanisms by which these single-point mutations exert an effect on the channel’s function and highlight the complexity of the multidomain channel, as well as the need for targeted therapies.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

scholarly journals Regulation of Melatonin 1a Receptor Signaling and Trafficking by Asparagine-124

2001 ◽  
Vol 15 (8) ◽  
pp. 1306-1317 ◽  
Author(s):  
Cole S. Nelson ◽  
Masayuki Ikeda ◽  
Heinrich S. Gompf ◽  
Mindi L. Robinson ◽  
Nadine K. Fuchs ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Point Mutations ◽  
Structural Effect ◽  
High Affinity ◽  
Intracellular Ca ◽  
Inhibitory Coupling ◽  
Trafficking Defects ◽  
Mutant Receptors ◽  
Camp Levels ◽  
Melatonin 1A Receptor

Abstract Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity 125I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K+-induced intracellular Ca++ fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind 125I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound 125I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca++ channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.

scholarly journals Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity

2017 ◽  
Vol 149 (2) ◽  
pp. 199-218 ◽  
Author(s):  
Akira Uehara ◽  
Takashi Murayama ◽  
Midori Yasukochi ◽  
Michael Fill ◽  
Minoru Horie ◽  
...  
Keyword(s):  
Single Channel ◽  
Single Point ◽  
Ryanodine Receptors ◽  
Point Mutations ◽  
Hek293 Cells ◽  
Polymorphic Ventricular Tachycardia ◽  
Single Point Mutation ◽  
Control Input ◽  
Life Threatening ◽  
Functional Consequences

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+ release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+ oscillations. An endoplasmic reticulum–targeted Ca2+ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+ leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+ leak from the sarcoplasmic reticulum, frequent Ca2+ waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

2019 ◽  
Author(s):  
Nobutaka Fujieda ◽  
Miho Yuasa ◽  
Yosuke Nishikawa ◽  
Genji Kurisu ◽  
Shinobu Itoh ◽  
...  
Keyword(s):  
Michael Addition ◽  
In Silico ◽  
Addition Reaction ◽  
Single Point ◽  
Point Mutations ◽  
Unsaturated Ketone ◽  
Michael Addition Reaction ◽  
Beta Barrel ◽  
Cupin Superfamily ◽  
Single Point Mutations

Cupin superfamily proteins (TM1459) work as a macromolecular ligand framework with a double-stranded beta-barrel structure ligating to a Cu ion through histidine side chains. Variegating the first coordination sphere of TM1459 revealed that H52A and H54A/H58A mutants effectively catalyzed the diastereo- and enantio-selective Michael addition reaction of nitroalkanes to an α,β-unsaturated ketone. Moreover, in silico substrate docking signified C106N and F104W single-point mutations, which inverted the diastereoselectivity of H52A and further improved the stereoselectivity of H54A/H58A, respectively.

Coaction of Electrostatic and Hydrophobic Interactions: Dynamic Constraints on Disordered TrkA Juxtamembrane Domain

2019 ◽  
Author(s):  
Zichen Wang ◽  
Huaxun Fan ◽  
Xiao Hu ◽  
John Khamo ◽  
Jiajie Diao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Function ◽  
Hydrophobic Interactions ◽  
Transmembrane Helix ◽  
Point Mutations ◽  
Salt Bridge ◽  
Receptor Activation ◽  
Membrane Dynamics ◽  
Juxtamembrane Domain ◽  
Joint Work

<p>The receptor tyrosine kinase family transmits signals into cell via a single transmembrane helix and a flexible juxtamembrane domain (JMD). Membrane dynamics makes it challenging to study the structural mechanism of receptor activation experimentally. In this study, we employ all-atom molecular dynamics with Highly Mobile Membrane-Mimetic to capture membrane interactions with the JMD of tropomyosin receptor kinase A (TrkA). We find that PIP<sub>2 </sub>lipids engage in lasting binding to multiple basic residues and compete with salt bridge within the peptide. We discover three residues insertion into the membrane, and perturb it through computationally designed point mutations. Single-molecule experiments indicate the contribution from hydrophobic insertion is comparable to electrostatic binding, and in-cell experiments show that enhanced TrkA-JMD insertion promotes receptor ubiquitination. Our joint work points to a scenario where basic and hydrophobic residues on disordered domains interact with lipid headgroups and tails, respectively, to restrain flexibility and potentially modulate protein function.</p>

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells

2021 ◽  
Author(s):  
Marisa L. Martino ◽  
Stephen N. Crooke ◽  
Marianne Manchester ◽  
M.G. Finn
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Virus Like Particles ◽  
Single Point Mutations

scholarly journals D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2622
Author(s):  
Romina Oliva ◽  
Abdul Rajjak Shaikh ◽  
Andrea Petta ◽  
Anna Vangone ◽  
Luigi Cavallo
Keyword(s):  
Three Dimensional ◽  
Salt Bridge ◽  
Structural Effect ◽  
Host Cells ◽  
Heptad Repeat ◽  
Genomic Sequences ◽  
Strong Interactions ◽  
Virus Infectivity ◽  
S Protein ◽  
Global Threat

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The “fusion core” of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.

scholarly journals mCSM-membrane: predicting the effects of mutations on transmembrane proteins

2020 ◽  
Vol 48 (W1) ◽  
pp. W147-W153 ◽  
Author(s):  
Douglas E V Pires ◽  
Carlos H M Rodrigues ◽  
David B Ascher
Keyword(s):  
Membrane Proteins ◽  
Single Point ◽  
Point Mutations ◽  
Web Server ◽  
Protein Coding ◽  
Pathogenic Variants ◽  
Model Protein ◽  
Matthew’S Correlation Coefficient ◽  
Membrane Protein Stability ◽  
User Friendly

Abstract Significant efforts have been invested into understanding and predicting the molecular consequences of mutations in protein coding regions, however nearly all approaches have been developed using globular, soluble proteins. These methods have been shown to poorly translate to studying the effects of mutations in membrane proteins. To fill this gap, here we report, mCSM-membrane, a user-friendly web server that can be used to analyse the impacts of mutations on membrane protein stability and the likelihood of them being disease associated. mCSM-membrane derives from our well-established mutation modelling approach that uses graph-based signatures to model protein geometry and physicochemical properties for supervised learning. Our stability predictor achieved correlations of up to 0.72 and 0.67 (on cross validation and blind tests, respectively), while our pathogenicity predictor achieved a Matthew's Correlation Coefficient (MCC) of up to 0.77 and 0.73, outperforming previously described methods in both predicting changes in stability and in identifying pathogenic variants. mCSM-membrane will be an invaluable and dedicated resource for investigating the effects of single-point mutations on membrane proteins through a freely available, user friendly web server at http://biosig.unimelb.edu.au/mcsm_membrane.

scholarly journals Sex-related effects on diabetes-induced alterations in calcium release in the rat heart

2007 ◽  
Vol 293 (6) ◽  
pp. H3584-H3592 ◽  
Author(s):  
Nazmi Yaras ◽  
Erkan Tuncay ◽  
Nuhan Purali ◽  
Babur Sahinoglu ◽  
Guy Vassort ◽  
...  
Keyword(s):  
Calcium Release ◽  
Binding Protein ◽  
Ryanodine Receptors ◽  
Left Ventricular ◽  
Adult Rats ◽  
Fk506 Binding Protein ◽  
Pressure Development ◽  
Spatiotemporal Parameters ◽  
Intracellular Ca ◽  
Developed Pressure

The present study was designed to determine whether the properties of local Ca2+ release and its related regulatory mechanisms might provide insight into the role of sex differences in heart functions of control and streptozotocin-induced diabetic adult rats. Left ventricular developed pressure, the rates of pressure development and decay (±dP/d t), basal intracellular Ca2+ level ([Ca2+]i), and spatiotemporal parameters of [Ca2+]i transients were found to be similar in male and female control rats. However, spatiotemporal parameters of Ca2+ sparks in cardiomyocytes isolated from control females were significantly larger and slower than those in control males. Diabetes reduced left ventricular developed pressure to a lower extent in females than in males, and the diabetes-induced depressions in both +dP/d t and −dP/d t were less in females than in males. Diabetes elicited a smaller reduction in the amplitude of [Ca2+]i transients in females than in males, a smaller reduction in sarcoplasmic reticulum-Ca2+ load, and less increase in basal [Ca2+]i. Similarly, the elementary Ca2+ events and their control proteins were clearly different in both sexes, and these differences were more marked in diabetes. Diabetes-induced depression of the Ca2+ spark amplitude was significantly less in females than in matched males. Levels of cardiac ryanodine receptors (RyR2) and FK506-binding protein 12.6 in control females were significantly higher than those shown in control males. Diabetes induced less RyR2 phosphorylation and FK506-binding protein 12.6 unbinding in females. Moreover, total and free sulfhydryl groups were significantly less reduced, and PKC levels were less increased, in diabetic females than in diabetic males. The present data related to local Ca2+ release and its related proteins describe some of the mechanisms that may underlie sex-related differences accounting for females to have less frequent development of cardiac diseases.

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