Conformational heterogeneity of the calmodulin binding interface

Abstract Calmodulin (CaM) is a ubiquitous Ca2+ sensor and a crucial signalling hub in many pathways aberrantly activated in disease. However, the mechanistic basis of its ability to bind diverse signalling molecules including G-protein-coupled receptors, ion channels and kinases remains poorly understood. Here we harness the high resolution of molecular dynamics simulations and the analytical power of Markov state models to dissect the molecular underpinnings of CaM binding diversity. Our computational model indicates that in the absence of Ca2+, sub-states in the folded ensemble of CaM’s C-terminal domain present chemically and sterically distinct topologies that may facilitate conformational selection. Furthermore, we find that local unfolding is off-pathway for the exchange process relevant for peptide binding, in contrast to prior hypotheses that unfolding might account for binding diversity. Finally, our model predicts a novel binding interface that is well-populated in the Ca2+-bound regime and, thus, a candidate for pharmacological intervention.

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Activation-triggered subunit exchange between CaMKII holoenzymes facilitates the spread of kinase activity

eLife ◽

10.7554/elife.01610 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 48

Author(s):

Margaret Stratton ◽

Il-Hyung Lee ◽

Moitrayee Bhattacharyya ◽

Sune M Christensen ◽

Luke H Chao ◽

...

Keyword(s):

Single Molecule ◽

Kinase Activity ◽

Exchange Process ◽

Memory Formation ◽

Information Storage ◽

Remarkable Property ◽

Subunit Exchange ◽

Calmodulin Binding ◽

Microscopy Technique ◽

Dynamics Simulations

The activation of the dodecameric Ca2+/calmodulin dependent kinase II (CaMKII) holoenzyme is critical for memory formation. We now report that CaMKII has a remarkable property, which is that activation of the holoenzyme triggers the exchange of subunits between holoenzymes, including unactivated ones, enabling the calcium-independent phosphorylation of new subunits. We show, using a single-molecule TIRF microscopy technique, that the exchange process is triggered by the activation of CaMKII, and that exchange is modulated by phosphorylation of two residues in the calmodulin-binding segment, Thr 305 and Thr 306. Based on these results, and on the analysis of molecular dynamics simulations, we suggest that the phosphorylated regulatory segment of CaMKII interacts with the central hub of the holoenzyme and weakens its integrity, thereby promoting exchange. Our results have implications for an earlier idea that subunit exchange in CaMKII may have relevance for information storage resulting from brief coincident stimuli during neuronal signaling.

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Allosteric modulation of GPCR-induced β-arrestin trafficking and signaling by a synthetic intrabody

10.1101/2022.01.11.475811 ◽

2022 ◽

Author(s):

Mithu Baidya ◽

Madhu Chaturvedi ◽

Hemlata Dwivedi-Agnihotri ◽

Ashutosh Ranjan ◽

Dominic Devost ◽

...

Keyword(s):

Plasma Membrane ◽

Phosphorylation Site ◽

Endosomal Trafficking ◽

Kinase Activation ◽

Primary Determinant ◽

Mechanistic Basis ◽

Dynamics Simulations ◽

Targeted Drug Discovery ◽

G Protein Coupled ◽

Current Paradigm

Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) is a primary determinant of β-arrestin (βarr) recruitment and trafficking. For several GPCRs, such as the vasopressin type II receptor (V2R), which exhibit high affinity for βarrs, agonist-stimulation first drives the translocation of βarrs to the plasma membrane, followed by endosomal trafficking. We previously found that mutation of a single phosphorylation site in V2R (i.e., V2RT360A) results in near-complete loss of βarr translocation to endosomes although βarrs are robustly recruited to the plasma membrane. Here, we show that a synthetic intrabody referred to as intrabody30 (Ib30), which selectively recognizes an active-like βarr1 conformation, rescues endosomal translocation of βarr1 for V2RT360A. In addition, Ib30 also rescues agonist-induced ERK1/2 MAP kinase activation for V2RT360A to levels similar to that of the wild-type V2R. Molecular dynamics simulations reveal that Ib30 binding promotes active-like conformation in βarr1 with respect to the inter-domain rotation. Interestingly, we also observe that Ib30 enhances the interaction of βarr1 with β2-adaptin, which provides a mechanistic basis for the ability of Ib30 to promote endosomal trafficking of βarr1. Taken together, our data provide a novel mechanism to positively modulate the receptor-transducer-effector axis for GPCRs using intrabodies, which can potentially be integrated in the current paradigm of GPCR-targeted drug discovery.

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Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics

Nature Communications ◽

10.1038/s41467-021-24674-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Bertrand Beckert ◽

Elodie C. Leroy ◽

Shanmugapriya Sothiselvam ◽

Lars V. Bock ◽

Maxim S. Svetlov ◽

...

Keyword(s):

Antibiotic Resistance Genes ◽

Structural Basis ◽

Sequence Motifs ◽

Specific Sequence ◽

Bacterial Ribosome ◽

Translational Arrest ◽

Exit Tunnel ◽

Mechanistic Basis ◽

Dynamics Simulations ◽

Context Specific

AbstractMacrolides and ketolides comprise a family of clinically important antibiotics that inhibit protein synthesis by binding within the exit tunnel of the bacterial ribosome. While these antibiotics are known to interrupt translation at specific sequence motifs, with ketolides predominantly stalling at Arg/Lys-X-Arg/Lys motifs and macrolides displaying a broader specificity, a structural basis for their context-specific action has been lacking. Here, we present structures of ribosomes arrested during the synthesis of an Arg-Leu-Arg sequence by the macrolide erythromycin (ERY) and the ketolide telithromycin (TEL). Together with deep mutagenesis and molecular dynamics simulations, the structures reveal how ERY and TEL interplay with the Arg-Leu-Arg motif to induce translational arrest and illuminate the basis for the less stringent sequence-specific action of ERY over TEL. Because programmed stalling at the Arg/Lys-X-Arg/Lys motifs is used to activate expression of antibiotic resistance genes, our study also provides important insights for future development of improved macrolide antibiotics.

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Local structure and distortions of mixed methane-carbon dioxide hydrates

Communications Chemistry ◽

10.1038/s42004-020-00441-7 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Bernadette R. Cladek ◽

S. Michelle Everett ◽

Marshall T. McDonnell ◽

Matthew G. Tucker ◽

David J. Keffer ◽

...

Keyword(s):

Carbon Dioxide ◽

Exchange Process ◽

Carbon Dioxide Sequestration ◽

Distribution Functions ◽

Atomic Scale ◽

Mixed Gas ◽

Pure Compounds ◽

Dynamics Simulations ◽

Pair Distribution Functions ◽

Methane Carbon

AbstractA vast source of methane is found in gas hydrate deposits, which form naturally dispersed throughout ocean sediments and arctic permafrost. Methane may be obtained from hydrates by exchange with hydrocarbon byproduct carbon dioxide. It is imperative for the development of safe methane extraction and carbon dioxide sequestration to understand how methane and carbon dioxide co-occupy the same hydrate structure. Pair distribution functions (PDFs) provide atomic-scale structural insight into intermolecular interactions in methane and carbon dioxide hydrates. We present experimental neutron PDFs of methane, carbon dioxide and mixed methane-carbon dioxide hydrates at 10 K analyzed with complementing classical molecular dynamics simulations and Reverse Monte Carlo fitting. Mixed hydrate, which forms during the exchange process, is more locally disordered than methane or carbon dioxide hydrates. The behavior of mixed gas species cannot be interpolated from properties of pure compounds, and PDF measurements provide important understanding of how the guest composition impacts overall order in the hydrate structure.

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Novel Dominant KCNQ2 Exon 7 Partial In-Frame Duplication in a Complex Epileptic and Neurodevelopmental Delay Syndrome

International Journal of Molecular Sciences ◽

10.3390/ijms21124447 ◽

2020 ◽

Vol 21 (12) ◽

pp. 4447

Author(s):

Pedro A. Lazo ◽

Juan L. García ◽

Paulino Gómez-Puertas ◽

Íñigo Marcos-Alcalde ◽

Cesar Arjona ◽

...

Keyword(s):

Protein Function ◽

De Novo ◽

3D Structure ◽

Unknown Origin ◽

Unknown Etiology ◽

Neurodevelopmental Delay ◽

Calmodulin Binding ◽

Whole Exome ◽

Dynamics Simulations ◽

Frame Duplication

Complex neurodevelopmental syndromes frequently have an unknown etiology, in which genetic factors play a pathogenic role. This study utilizes whole-exome sequencing (WES) to examine four members of a family with a son presenting, since birth, with epileptic-like crises, combined with cerebral palsy, severe neuromotor and developmental delay, dystonic tetraparexia, axonal motor affectation, and hyper-excitability of unknown origin. The WES study detected within the patient a de novo heterozygous in-frame duplication of thirty-six nucleotides within exon 7 of the human KCNQ2 gene. This insertion duplicates the first twelve amino acids of the calmodulin binding site I. Molecular dynamics simulations of this KCNQ2 peptide duplication, modelled on the 3D structure of the KCNQ2 protein, suggest that the duplication may lead to the dysregulation of calcium inhibition of this protein function.

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MECHANISMS IN ENDOCRINOLOGY: An update in the genetic aetiologies of combined pituitary hormone deficiency

Acta Endocrinologica ◽

10.1530/eje-15-1095 ◽

2016 ◽

Vol 174 (6) ◽

pp. R239-R247 ◽

Cited By ~ 26

Author(s):

Frederic Castinetti ◽

Rachel Reynaud ◽

Alexandru Saveanu ◽

Nicolas Jullien ◽

Marie Helene Quentien ◽

...

Keyword(s):

Transcription Factors ◽

G Protein Coupled Receptors ◽

Hormone Deficiency ◽

Immunoglobulin Superfamily ◽

Pituitary Hormone ◽

Pituitary Hormone Deficiency ◽

Combined Pituitary Hormone Deficiency ◽

Signalling Molecules ◽

Genes Encoding ◽

G Protein Coupled

Over the last 5 years, new actors involved in the pathogenesis of combined pituitary hormone deficiency in humans have been reported: they included a member of the immunoglobulin superfamily glycoprotein and ciliary G protein-coupled receptors, as well as new transcription factors and signalling molecules. New modes of inheritance for alterations of genes encoding transcription factors have also been described. Finally, actors known to be involved in a very specific phenotype (hypogonadotroph hypogonadism for instance) have been identified in a wider range of phenotypes. These data thus suggest that new mechanisms could explain the low rate of aetiological identification in this heterogeneous group of diseases. Taking into account the fact that several reviews have been published in recent years on classical aetiologies of CPHD such as mutations ofPOU1F1orPROP1, we focused the present overview on the data published in the last 5 years, to provide the reader with an updated review on this rapidly evolving field of knowledge.

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The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface

PLoS Pathogens ◽

10.1371/journal.ppat.1009368 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009368

Author(s):

Juan Carlos De la Concepcion ◽

Josephine H. R. Maidment ◽

Apinya Longya ◽

Gui Xiao ◽

Marina Franceschetti ◽

...

Keyword(s):

Molecular Mechanisms ◽

Allelic Variation ◽

Immune Recognition ◽

In Planta ◽

Pathogen Effectors ◽

Binding Interface ◽

Recognition Specificity ◽

Blast Fungus ◽

Resistance Specificity ◽

Mechanistic Basis

Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.

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Constitutive signal bias mediated by the human GHRHR splice variant 1

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2106606118 ◽

2021 ◽

Vol 118 (40) ◽

pp. e2106606118

Author(s):

Zhaotong Cong ◽

Fulai Zhou ◽

Chao Zhang ◽

Xinyu Zou ◽

Huibing Zhang ◽

...

Keyword(s):

Splice Variant ◽

Human Growth ◽

Bound State ◽

G Protein Coupled Receptors ◽

Cancer Cell Proliferation ◽

Downstream Signaling ◽

Functional Studies ◽

Dynamics Simulations ◽

Protein Molecular Dynamics ◽

G Protein Coupled

Alternative splicing of G protein–coupled receptors has been observed, but their functions are largely unknown. Here, we report that a splice variant (SV1) of the human growth hormone–releasing hormone receptor (GHRHR) is capable of transducing biased signal. Differing only at the receptor N terminus, GHRHR predominantly activates Gs while SV1 selectively couples to β-arrestins. Based on the cryogenic electron microscopy structures of SV1 in the apo state or GHRH-bound state in complex with the Gs protein, molecular dynamics simulations reveal that the N termini of GHRHR and SV1 differentiate the downstream signaling pathways, Gs versus β-arrestins. As suggested by mutagenesis and functional studies, it appears that GHRH-elicited signal bias toward β-arrestin recruitment is constitutively mediated by SV1. The level of SV1 expression in prostate cancer cells is also positively correlated with ERK1/2 phosphorylation but negatively correlated with cAMP response. Our findings imply that constitutive signal bias may be a mechanism that ensures cancer cell proliferation.

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5-Lipoxygenase Pathway, Dendritic Cells, and Adaptive Immunity

Journal of Biomedicine and Biotechnology ◽

10.1155/s1110724304310041 ◽

2004 ◽

Vol 2004 (2) ◽

pp. 99-105 ◽

Cited By ~ 33

Author(s):

Harizi Hedi ◽

Gualde Norbert

Keyword(s):

Dendritic Cells ◽

Adaptive Immunity ◽

Inflammatory Cells ◽

Lipid Mediators ◽

G Protein Coupled Receptors ◽

Lipoxygenase Pathway ◽

Innate And Adaptive Immunity ◽

Paracrine Signalling ◽

Signalling Molecules ◽

G Protein Coupled

5-lipoxygenase (5-LO) pathway is the major source of potent proinflammatory leukotrienes (LTs) issued from the metabolism of arachidonic acid (AA), and best known for their roles in the pathogenesis of asthma. These lipid mediators are mainly released from myeloid cells and may act as physiological autocrine and paracrine signalling molecules, and play a central role in regulating the interaction between innate and adaptive immunity. The biological actions of LTs including their immunoregulatory and proinflammatory effects are mediated through extracellular specific G-protein-coupled receptors. Despite their role in inflammatory cells, such as neutrophils and macrophages, LTs may have important effects on dendritic cells (DC)-mediated adaptive immunity. Several lines of evidence show that DC not only are important source of LTs, but also become targets of their actions by producing other lipid mediators and proinflammatory molecules. This review focuses on advances in 5-LO pathway biology, the production of LTs from DC and their role on various cells of immune system and in adaptive immunity.

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