Structural Basis for Allosteric Control of the SERCA-Phospholamban Membrane Complex by Ca2+ and cAMP-dependent Phosphorylation

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight- or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme’s function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN’s cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme’s active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

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Structural Basis for Allosteric Control of the SERCA-Phospholamban Membrane Complex by Ca2+ and Phosphorylation

eLife ◽

10.7554/elife.66226 ◽

2021 ◽

Vol 10 ◽

Author(s):

Gianluigi Veglia ◽

Daniel K Weber ◽

Venkateswara U Reddy ◽

Songlin Wang ◽

Erik K Larsen ◽

...

Keyword(s):

Structural Basis ◽

Adrenergic Stimulation ◽

Structural Refinement ◽

Oriented Sample ◽

Membrane Complex ◽

Binding Interface ◽

Plasmic Reticulum ◽

Transport Response ◽

Dependent Protein ◽

Fight Or Flight Response

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport response to β-adrenergic stimulation, thus modulating cardiac output during the fight-or-flight response. In the sarcoplasmic reticulum membrane, PLN binds to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca2+ concentration reverses the inhibitory effects through an unknown mechanism. Using oriented-sample solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we reveal that phosphorylation of PLN’s cytoplasmic regulatory domain signals the disruption of several inhibitory contacts at the transmembrane binding interface of the SERCA-PLN complex that are propagated to the enzyme’s active site, augmenting Ca2+ transport. Our findings address long-standing questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally-modified bitopic membrane proteins.

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Visualization of Head–Head Interactions in the Inhibited State of Smooth Muscle Myosin

The Journal of Cell Biology ◽

10.1083/jcb.147.7.1385 ◽

1999 ◽

Vol 147 (7) ◽

pp. 1385-1390 ◽

Cited By ~ 84

Author(s):

Thomas Wendt ◽

Dianne Taylor ◽

Terri Messier ◽

Kathleen M. Trybus ◽

Kenneth A. Taylor

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Myosin ◽

Intramolecular Interactions ◽

Actin Binding ◽

Structural Basis ◽

Binding Interface ◽

Structural Differences ◽

Muscle Myosin ◽

Positively Charged ◽

Phosphoryla Tion

The structural basis for the phosphoryla- tion-dependent regulation of smooth muscle myosin ATPase activity was investigated by forming two- dimensional (2-D) crystalline arrays of expressed unphosphorylated and thiophosphorylated smooth muscle heavy meromyosin (HMM) on positively charged lipid monolayers. A comparison of averaged 2-D projections of both forms at 2.3-nm resolution reveals distinct structural differences. In the active, thiophosphorylated form, the two heads of HMM interact intermolecularly with adjacent molecules. In the unphosphorylated or inhibited state, intramolecular interactions position the actin-binding interface of one head onto the converter domain of the second head, thus providing a mechanism whereby the activity of both heads could be inhibited.

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Structural basis of cyclic nucleotide selectivity in cGMP dependent protein kinase II

BMC Pharmacology and Toxicology ◽

10.1186/2050-6511-16-s1-a15 ◽

2015 ◽

Vol 16 (S1) ◽

Author(s):

James C. Campbell ◽

Kevin Y. Li ◽

Jeong Joo Kim ◽

Gilbert Huang ◽

Albert S. Reger ◽

...

Keyword(s):

Protein Kinase ◽

Cyclic Nucleotide ◽

Structural Basis ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Protein Kinase Ii ◽

Dependent Protein

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Structural basis for recognition of human 7SK long noncoding RNA by the La-related protein Larp7

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1806276115 ◽

2018 ◽

Vol 115 (28) ◽

pp. E6457-E6466 ◽

Cited By ~ 20

Author(s):

Catherine D. Eichhorn ◽

Yuan Yang ◽

Lucas Repeta ◽

Juli Feigon

Keyword(s):

Noncoding Rna ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcription Elongation ◽

Structural Basis ◽

Titration Calorimetry ◽

Rna Recognition Motif ◽

Related Protein ◽

Binding Interface ◽

And Function

The La and the La-related protein (LARP) superfamily is a diverse class of RNA binding proteins involved in RNA processing, folding, and function. Larp7 binds to the abundant long noncoding 7SK RNA and is required for 7SK ribonucleoprotein (RNP) assembly and function. The 7SK RNP sequesters a pool of the positive transcription elongation factor b (P-TEFb) in an inactive state; on release, P-TEFb phosphorylates RNA Polymerase II to stimulate transcription elongation. Despite its essential role in transcription, limited structural information is available for the 7SK RNP, particularly for protein–RNA interactions. Larp7 contains an N-terminal La module that binds UUU-3′OH and a C-terminal atypical RNA recognition motif (xRRM) required for specific binding to 7SK and P-TEFb assembly. Deletion of the xRRM is linked to gastric cancer in humans. We report the 2.2-Å X-ray crystal structure of the human La-related protein group 7 (hLarp7) xRRM bound to the 7SK stem-loop 4, revealing a unique binding interface. Contributions of observed interactions to binding affinity were investigated by mutagenesis and isothermal titration calorimetry. NMR 13C spin relaxation data and comparison of free xRRM, RNA, and xRRM–RNA structures show that the xRRM is preordered to bind a flexible loop 4. Combining structures of the hLarp7 La module and the xRRM–7SK complex presented here, we propose a structural model for Larp7 binding to the 7SK 3′ end and mechanism for 7SK RNP assembly. This work provides insight into how this domain contributes to 7SK recognition and assembly of the core 7SK RNP.

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Abstract 1078: β-Adrenergic Stimulation of Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) Overexpressing Myocytes Increases Sarcoplasmic Reticulum (SR) Calcium Leak Causing KN-93 Sensitive Arrhythmias

Circulation ◽

10.1161/circ.116.suppl_16.ii_216 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Daniel P Wadsack ◽

Michael Kohlhaas ◽

Adam G Rokita ◽

Stefan Neef ◽

Lars S Maier

Keyword(s):

Control Level ◽

Adrenergic Stimulation ◽

Dependent Protein Kinase ◽

Inotropic Effects ◽

Positive Inotropic Effects ◽

Intracellular Ca ◽

Dependent Protein ◽

Ca Homeostasis ◽

Sr Calcium Leak ◽

Inotropic Stimulation

CaMKII is associated with hypertrophy, heart failure and alters intracellular Ca homeostasis. An increased SR Ca leak due to phosphorylation of SR Ca release channels by CaMKII leads to decreased SR Ca content and impaired contractility. This loss of Ca from the SR may also contribute to arrhythmias. We investigated whether β-adrenergic stimulation with isoproterenol (ISO) normalizes SR Ca content and whether inhibiting CaMKII reduces arrhythmias. CaMKII-overexpressing rabbit and mouse myocytes were investigated. Cell shortening, Ca fluorescence (fluo-3) and the incidence of arrhythmias were assessed. An arrhythmia-score differentiated between: early-spike-arrhythmias (ESA), late-spike-arrhythmias (LSA) and permanent arrhythmias (PA). ISO (37°C) had significantly different effects on myocytes with acute (24 h, rabbit, n=34) or chronic (22 w, mouse, n=34) CaMKII overexpression vs corresponding control myocytes (LacZ, n=21 or WT n=34). CaMKII overexpression lead to an ISO concentration-dependent (10 −10 -10 −5 mol/L) inotropic but compared to WT (or LacZ, respectively) impaired shortening and Ca transients (two-way ANOVA, P <0.05). A similar difference between CaMKII-overexpressing (n=17) and WT (n=19) myocytes was also seen during a shortening-frequency protocol (stepwise increase from 0.1– 4 Hz, two-way ANOVA, P <0.05). Arrhythmias spontaneously occurred in CaMKII-overexpressing mouse myocytes. With β-inotropic stimulation (10 −6 mol/L ISO) arrhythmias were increased 6.4-fold. Appearance of ESA and PA could be significantly reduced by KN-93 (1 μmol/L). At a basal stimulation rate of 1 Hz and 10 −7 mol/L ISO, PA could be dramatically reduced by half from control-level 21.43% (KN-92, inactive derivative, n=42) down to 10.87% (KN-93, n=46) arrhythmic events. ESA could be reduced almost 4-fold from 16.67% (KN-92) to 4.35% in the presence of KN-93. We conclude from these data that increasing ISO concentrations exerts positive inotropic effects but cannot normalize altered Ca handling in CaMKII-overexpressing myocytes. This may be due to an increased SR Ca leak under these conditions thus contributing to the arrhythmias observed. CaMKII inhibition clearly can reduce arrhythmias in the presence of β-adrenergic stimulation with ISO.

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Structural basis for auxiliary subunit KCTD16 regulation of the GABABreceptor

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1903024116 ◽

2019 ◽

Vol 116 (17) ◽

pp. 8370-8379 ◽

Cited By ~ 11

Author(s):

Hao Zuo ◽

Ian Glaaser ◽

Yulin Zhao ◽

Igor Kurinov ◽

Lidia Mosyak ◽

...

Keyword(s):

Structural Basis ◽

Functional Coupling ◽

Girk Channels ◽

Receptor Complexes ◽

Binding Interface ◽

Tetramerization Domain ◽

High Resolution Structure ◽

Inwardly Rectifying ◽

The Brain ◽

Oligomerization Domain

Metabotropic GABABreceptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABABreceptor complexes contain the principal subunits GABAB1and GABAB2, which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABABreceptors and modify the kinetics of GABABreceptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABABreceptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABAB2receptor. A single GABAB2C-terminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABAB2–KCTD16 interface disrupted both the biochemical association and functional modulation of GABABreceptors and G protein-activated inwardly rectifying K+channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABABreceptors. Defining the binding interface of GABABreceptor and KCTD reveals a potential regulatory site for modulating GABAB-receptor function in the brain.

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Characterization of the Retinoid Orphan-Related Receptor-α Coactivator Binding Interface: A Structural Basis for Ligand-Independent Transcription

Molecular Endocrinology ◽

10.1210/mend.16.5.0837 ◽

2002 ◽

Vol 16 (5) ◽

pp. 998-1012 ◽

Cited By ~ 1

Author(s):

Jonathan M. Harris ◽

Patrick Lau ◽

Shen Liang Chen ◽

George E. O. Muscat

Keyword(s):

Structural Basis ◽

Binding Interface

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Digital transcriptome analysis indicates adaptive mechanisms in the heart of a hibernating mammal

Physiological Genomics ◽

10.1152/physiolgenomics.00076.2005 ◽

2005 ◽

Vol 23 (2) ◽

pp. 227-234 ◽

Cited By ~ 66

Author(s):

Katharine M. Brauch ◽

Nirish D. Dhruv ◽

Eric A. Hanse ◽

Matthew T. Andrews

Keyword(s):

High Throughput Sequencing ◽

Digital Gene Expression ◽

Ground Squirrels ◽

Pyruvate Dehydrogenase Kinase ◽

Cdna Libraries ◽

Body Temperatures ◽

Triacylglycerol Lipase ◽

Plasmic Reticulum ◽

Comprehensive Survey ◽

Dependent Protein

Survival of near-freezing body temperatures and reduced blood flow during hibernation is likely the result of changes in the expression of specific genes. In this study, we described a comprehensive survey of mRNAs in the heart of the thirteen-lined ground squirrel ( Spermophilus tridecemlineatus) before and during hibernation. The heart was chosen for this study because it is a contractile organ that must continue to work despite body temperatures of 5°C and the lack of food for periods of 5–6 mo. We used a digital gene expression assay involving high-throughput sequencing of directional cDNA libraries from hearts of active and hibernating ground squirrels to determine the identity and frequency of 3,532 expressed sequence tags (ESTs). Statistical analysis of the active and hibernating heart expression profile indicated the differential regulation of 48 genes based on a P ≤ 0.03 threshold. Several of the differentially expressed genes identified in this screen encode proteins that likely account for uninterrupted cardiac function during hibernation, including those involved in metabolism, contractility, Ca2+ handling, and low-temperature catalysis. A sampling of genes showing higher expression during hibernation includes phosphofructokinase, pancreatic triacylglycerol lipase, pyruvate dehydrogenase kinase 4 (PDK4), aldolase A, sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), titin, and four-and-a-half LIM domains protein 2 (FHL2). Genes showing reduced levels of expression during hibernation include cyclin-dependent kinase 2-associated protein 1 (CDK2AP1), troponin C, phospholamban, Ca2+/calmodulin-dependent protein kinase II (CaMKII), calmodulin, and four subunits of cytochrome c oxidase.

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Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes

Blood ◽

10.1182/blood.v122.21.2358.2358 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2358-2358

Author(s):

Justin D Walter ◽

Rachel A Werther ◽

Caileen M Brison ◽

John F. Healey ◽

Shannon L. Meeks ◽

...

Keyword(s):

Factor Viii ◽

Ternary Complex ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Structural Basis ◽

C2 Domain ◽

Proteolytic Activation ◽

X Ray ◽

Binding Interface ◽

Inhibitory Antibodies

Abstract The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies. Disclosures: No relevant conflicts of interest to declare.

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