Papers by Gianluigi Veglia
Frequency-selective heteronuclear dephasing by dipole couplings in spinning and static solids
The Journal of Chemical Physics, 1996
A compensated pulse sequence for the spectrally selective reintroduction of heteronuclear dipole–... more A compensated pulse sequence for the spectrally selective reintroduction of heteronuclear dipole–dipole interactions (frequency-selective dipolar recoupling) into solid state magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments is described and shown to provide frequency-selective dipolar dephasing in weakly coupled spin systems. The experimental dipolar spin evolution is interpreted via analytical and numerical calculations, which include a simple model for the observed losses of spin coherence in the multiple pulse experiments. In the peptide glycylglycine, the selective dipolar evolution of two spins is observed while the influence of larger internuclear couplings is suppressed. This approach is aimed at obtaining several quantitative internuclear distances independently in dipolar ‘‘recoupling’’ MAS experiments by reducing multiple spin effects in the observed dipolar evolution. Similar frequency-selective dephasing experiments are also introduced for static s...
Chemical Reviews, 2018
Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanism... more Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.
eLife, 2021
Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca 2+ -transpor... more Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca 2+ -transport response to b-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 Ca 2+ -ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca 2+ concentration reverses the inhibitory effects through an unknown mechanism. Using orientedsample 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 Ca 2+ transport. Our findings address longstanding questions about SERCA regulation, epitomizing a signal transduction mechanism operated by posttranslationally modified bitopic membrane proteins.
bioRxiv, 2020
Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport ... more 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 enzyme9s function within a narrow physiological window. PLN phosphorylation or increase in Ca2+ concentration reverses the inhibitory effects. Despite a plethora of X-ray studies, the structural mechanism of SERCA regulation by PLN remains unknown. Using solid-state NMR spectroscopy and replica-averaged NMR-restrained structural refinement, we found that the transmembrane region of PLN is in equilibrium between inhibitory and non-inhibitory topologies within SERCA9s binding groove. Phosphorylation of PLN at Ser16, or increase in Ca2+ concentration, shifts the equilibrium toward the non-inhibitory topologies, augmenting Ca2+ transport and muscle contractility. This t...
Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca2+-transport ... more 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 tran...
Methods, 2018
The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of... more The intrinsic conformational plasticity of membrane proteins directly influences the magnitude of the orientational-dependent NMR interactions such as dipolar couplings (DC) and chemical shift anisotropy (CSA). As a result, the conventional cross-polarization (CP)-based techniques mainly capture the more rigid regions of membrane proteins, while the most dynamic regions are essentially invisible. Nonetheless, dynamic regions can be detected using experiments in which polarization transfer takes place via J-coupling interactions. Here, we review our recent efforts to develop single and dual acquisition pulse sequences with either 1 H or 13 C detection that utilize both DC and J-coupling mediated transfer to detect both rigid and mobile regions of membrane proteins in native-like lipid environments. We show the application of these new methods for studying the conformational equilibrium of a single-pass membrane protein, phospholamban, which regulates the calcium transport across the sarcoplasmic reticulum (SR) membrane by interacting with the SR Ca 2+ -ATPase. We anticipate that these methods will be ideal to portray the complex dynamics of membrane proteins in their native environments.
Biochimica et biophysica acta, Jun 1, 2018
Approximately, 70% of the Caion transport into the sarcoplasmic reticulum is catalyzed by the sar... more Approximately, 70% of the Caion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Cabinding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spe...
Scientific reports, Jan 15, 2016
The structural dynamics governing collective motions in oligomeric membrane proteins play key rol... more The structural dynamics governing collective motions in oligomeric membrane proteins play key roles in vital biomolecular processes at cellular membranes. In this study, we present a structural refinement approach that combines solid-state NMR experiments and molecular simulations to accurately describe concerted conformational transitions identifying the overall structural, dynamical, and topological states of oligomeric membrane proteins. The accuracy of the structural ensembles generated with this method is shown to reach the statistical error limit, and is further demonstrated by correctly reproducing orthogonal NMR data. We demonstrate the accuracy of this approach by characterising the pentameric state of phospholamban, a key player in the regulation of calcium uptake in the sarcoplasmic reticulum, and by probing its dynamical activation upon phosphorylation. Our results underline the importance of using an ensemble approach to characterise the conformational transitions that ...
Biophysical journal, Jan 17, 2014
Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology,... more Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR pa...
Methods in molecular biology (Clifton, N.J.), 2010
This chapter reviews the molecular biology, biochemical, and NMR methods that we used to study th... more This chapter reviews the molecular biology, biochemical, and NMR methods that we used to study the structural dynamics, membrane topology, and interaction of phospholamban (PLN), a small regulatory membrane protein involved in the regulation of the sarcoplasmic reticulum Ca-ATPase (SERCA). In particular, we show the progression of our research from the initial hypotheses toward understanding the molecular mechanisms of SERCA's regulation, including the effects of PLN oligomerization and posttranslational phosphorylation. Finally, we show how the knowledge of the molecular mechanism of the structural dynamics and topology of free and bound proteins can lead to the rational design of PLN analogs for possible use in gene therapy.
Biochimica et biophysica acta, 2015
Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic retic... more Phospholamban (PLN) is a single-pass membrane protein that regulates the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA). Phosphorylation of PLN at Ser16 reverses its inhibitory function under β-adrenergic stimulation, augmenting Ca²⁺ uptake in the sarcoplasmic reticulum and muscle contractility. PLN exists in two conformations; a T state, where the cytoplasmic domain is helical and adsorbed on the membrane surface, and an R state, where the cytoplasmic domain is unfolded and membrane detached. Previous studies have shown that the PLN conformational equilibrium is crucial to SERCA regulation. Here, we used a combination of solution and solid-state NMR to compare the structural topology and conformational dynamics of monomeric PLN (PLN(AFA)) with that of the PLN(R14del), a naturally occurring deletion mutant that is linked to the progression of dilated cardiomyopathy. We found that the behavior of the inhibitory transmembrane domain of PLN(R14del) is similar to that of the native se...
Advances in protein chemistry and structural biology, 2012
The catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is an exquisite example of a sin... more The catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is an exquisite example of a single molecule allosteric enzyme, where classical and modern views of allosteric signaling merge. In this chapter, we describe the mapping of PKA-C conformational dynamics and allosteric signaling in the free and bound states using a combination of NMR spectroscopy and molecular dynamics simulations. We show that ligand binding affects the enzyme's conformational dynamics, shaping the free-energy landscape toward the next stage of the catalytic cycle. While nucleotide and substrate binding enhance the enzyme's conformational entropy and define dynamically committed states, inhibitor binding attenuates the internal dynamics in favor of enthalpic interactions and delineates dynamically quenched states. These studies support a central role of conformational dynamics in many aspects of enzymatic turnover and suggest future avenues for controlling enzymatic function.
Biophysical Journal, 2015
The FEBS Journal, 2013
Protein kinase A (PKA) is the archetypical phosphokinase, sharing a catalytic core with the entir... more Protein kinase A (PKA) is the archetypical phosphokinase, sharing a catalytic core with the entire protein kinase superfamily. In eukaryotes, the ubiquitous location of PKA makes it one of the most important cellular signaling molecules, involved in a myriad of events. The catalytic subunit of PKA (PKA‐C) is one of the most studied enzymes and was the first kinase to be crystallized; however, the effects of ligand binding, post‐translational modifications and mutations on the activity of the kinase have been difficult to understand with only structural data. Here, we review our latest NMR studies on PKA‐C, the results of which underscore the role of fast and slow conformational dynamics in the activation and inhibition of the kinase.
Structure, 2013
Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), thereby regu... more Phospholamban (PLN) inhibits the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), thereby regulating cardiac diastole. In membranes, PLN assembles into homopentamers that in both the phosphorylated and nonphosphorylated states have been proposed to form ion-selective channels. Here, we determined the structure of the phosphorylated pentamer using a combination of solution and solid-state nuclear magnetic resonance methods. We found that the pinwheel architecture of the homopentamer is preserved upon phosphorylation, with each monomer having an L-shaped conformation. The TM domains form a hydrophobic pore approximately 24 A ˚long and 2 A ˚in diameter, which is inconsistent with canonical Ca 2+ -selective channels. Phosphorylation, however, enhances the conformational dynamics of the cytoplasmic region of PLN, causing partial unwinding of the amphipathic helix. We propose that PLN oligomers act as storage for active monomers, keeping SERCA function within a physiological window.
Proceedings of the National Academy of Sciences, 2007
Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(end... more Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: ( i ) extended from the bilayer normal by 50–60°; ( ii ) continuous α-helix tilted 28° relative to the bilayer normal; ( iii ) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and ( iv ) bellflower structure, in which the cytoplasmic domain helix makes ≈20° angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combinati...
Proceedings of the National Academy of Sciences, 2009
Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric... more Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein...
Proceedings of the National Academy of Sciences, 2013
Significance The sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)/phospholamban complex regulates car... more Significance The sarcoplasmic reticulum Ca 2+ -ATPase (SERCA)/phospholamban complex regulates cardiac muscle contractility by controlling Ca 2+ transport from the cytosol to the lumen of the sarcoplasmic reticulum. By mapping the interactions between these two membrane proteins, we found that SERCA function depends on the equilibria between transient conformational states of phospholamban. Phosphorylation of phospholamban shifts the equilibria, enhancing SERCA function. This mechanism explains why tuning phospholamban’s structural dynamics can modulate SERCA function and may aid in designing innovative therapeutic approaches to heart failure.
Proceedings of the National Academy of Sciences, 2011
Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2+ ... more Phospholamban (PLN) is a type II membrane protein that inhibits the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), thereby regulating calcium homeostasis in cardiac muscle. In membranes, PLN forms pentamers that have been proposed to function either as a storage for active monomers or as ion channels. Here, we report the T-state structure of pentameric PLN solved by a hybrid solution and solid-state NMR method. In lipid bilayers, PLN adopts a pinwheel topology with a narrow hydrophobic pore, which excludes ion transport. In the T state, the cytoplasmic amphipathic helices (domains Ia) are absorbed into the lipid bilayer with the transmembrane domains arranged in a left-handed coiled-coil configuration, crossing the bilayer with a tilt angle of approximately 11° with respect to the membrane normal. The tilt angle difference between the monomer and pentamer is approximately 13°, showing that intramembrane helix–helix association forces dominate over the hydrophobic mismatch, driving th...
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Papers by Gianluigi Veglia