Current Projects in the Veglia Lab

Cyclic AMP (cAMP) mediated cell signaling

A primary cAMP receptor is the cAMP-dependent protein kinase A (PKA). This enzyme was the first kinase to be crystallized and has been used as the prototypical example for the AGC protein kinase family. In the inactive form, PKA adopts a homotetrameric assembly (holoenzyme) with two catalytic subunits (C subunits or PKA-C) and two regulatory subunits (R subunits). The holoenzyme is anchored to the membrane via A-kinase anchoring protein (AKAP). The classical activation mechanism involves cAMP binding to the R subunits and the release of PKA-C, which is free to phosphorylate a plethora of substrates. While several aberrant mutations have been discovered in the R subunit, in the past decades, mutations, deletions, and fusions have been found in the PRKACA gene encoding for PKA-C. These modifications are responsible for dysregulating cAMP signaling and the progression of diseases such as Cushing’s syndrome, myxomas, and fibrolamellar hepatocellular carcinomas. Our group utilizes spectroscopic and biophysical methods to understand how these mutations perturb the structural dynamics and allosteric signaling of PKA-C, resulting in dysfunctional cAMP signaling and leading to disease.       

Selected publications

Olivieri et al. ATP-competitive inhibitors modulate the substrate binding cooperativity of a kinase by altering its conformational entropy. Sci Adv. 2022 Jul 29;8(30):eabo0696. doi: 10.1126/sciadv.abo0696. Epub 2022 Jul 29. PMID: 35905186; PMCID: PMC9337769.

Olivieri et al. Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A. Elife. 2020 Apr 27;9:e55607. doi: 10.7554/eLife.55607. PMID: 32338601; PMCID: PMC7234811.

Olivieri et al. Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A. Commun Biol. 2021 Mar 10;4(1):321. doi: 10.1038/s42003-021-01819-6.

Walker et al. Cushing's syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity. Sci Adv. 2019 Aug 28;5(8):eaaw9298. doi: 10.1126/sciadv.aaw9298. PMID: 31489371; PMCID: PMC6713507.

Wang et al. Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase's enzymatic cycle. Nat Commun. 2019 Feb 18;10(1):799. doi: 10.1038/s41467-019-08655-7. PMID: 30778078; PMCID: PMC6379427.



Calcium transport in skeletal and heart muscle

Calcium transport is central to cardiac and skeletal muscle contractility. Its homeostatic balance is modulated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA), which handles ~70% of intracellular Ca2+ regulation in humans. SERCA is an integral membrane protein whose function is regulated by an array of single-pass membrane proteins called regulins. Regulins keep this ATPase’s activity within a narrow physiological window. Dysregulation of SERCA activity degenerates into muscle disease. So far, seven regulins have been sequenced: phospholamban (PLN), sarcolipin (SLN), endoregulin (ELN), another regulin (ALN), myoregulin (MLN), dwarf open reading frame (DWORF), and sarcolamban (SCL, Drosophila m.). These regulins are single-pass membrane proteins that bind SERCA in the transmembrane domain and allosterically control SERCA’s apparent affinity for Ca2+ ions. Some regulins are post-translationally modified (e.g., phosphorylated, lipidation, acetylation, etc.). These events reverse or augment regulins’ regulatory function. In the past decade, it was found that SERCA’s regulatome has a new player, HAX-1, an intrinsically disordered protein that interacts with the other regulins to enhance their function. We aim to understand how regulins and HAX-1 interact with SERCA to augment or decrease Ca2+ transport and concomitant muscle contractility. Understanding the molecular determinant for Ca2+ transport by SERCA is critical to devise new and innovative therapy to counteract muscle disease, including heart failure.

Selected publications

Reddy et al. A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca2+-ATPase. Structure. 2022 Mar 3;30(3):360-370.e6. doi: 10.1016/j.str.2021.11.003. Epub 2021 Dec 6. PMID: 34875216; PMCID: PMC8897251.

Wang et al. Structural basis for sarcolipin's regulation of muscle thermogenesis by the sarcoplasmic reticulum Ca2+-ATPase. Sci Adv. 2021 Nov 26;7(48):eabi7154. doi: 10.1126/sciadv.abi7154. Epub 2021 Nov 26. PMID: 34826239; PMCID: PMC8626070.

Weber et al. Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation. Elife. 2021 May 12;10:e66226. doi: 10.7554/eLife.66226. PMID: 33978571; PMCID: PMC8184213.

Gopinath T et al. Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin? Acc Chem Res. 2021 Mar 16;54(6):1430-1439. doi: 10.1021/acs.accounts.0c00670. Epub 2021 Mar 3. PMID: 33655754.

Larsen et al. Intrinsically disordered HAX-1 regulates Ca2+ cycling by interacting with lipid membranes and the phospholamban cytoplasmic region. Biochim Biophys Acta Biomembr. 2020 Jan 1;1862(1):183034. doi: 10.1016/j.bbamem.2019.183034. Epub 2019 Aug 7. PMID: 31400305; PMCID: PMC6899184.


NMR methods development

Many of our projects necessitate the development of novel techniques to investigate the structure and dynamic interactions of these large protein complexes. Therefore, we dedicate a significant effort to developing new methods to improve solution- and solid-state NMR spectroscopy. Specifically, we have been pioneering methods for multiple acquisitions of solid-state NMR spectra utilizing orphan spin operators. These polarization optimized experiments (POE) constitute the basis for speeding up multidimensional solid-state NMR experiments and can be applied to static and magic angle spinning experiments of soluble and membrane-bound proteins. Currently, we are applying evolutionary algorithms and artificial intelligence to redesign RF pulses and pulse sequences to improve sensitivity and performance and upgrade them for applications of NMR spectroscopy at high- and ultra-high magnetic fields. We developed a new software (GENETICS-AI) to design high-fidelity RF shapes that are highly compensated for inhomogeneity for possible MR spectroscopy and imaging applications.       

Selected publications/patents

Patent : Gianluigi Veglia, Manu Veliparambil Subrahmanian. System and method for producing radiofrequency pulses in magnetic resonance using an optimal phase surface. US16/861,506,!/applications/16861506

Subrahmanian et al. High-fidelity control of spin ensemble dynamics via artificial intelligence: from quantum computing to NMR spectroscopy and imaging. PNAS Nexus. 2022 Aug 5;1(4):pgac133. doi: 10.1093/pnasnexus/pgac133. PMID: 36106184; PMCID: PMC9463062.

Manu VS et al. Water irradiation devoid pulses enhance the sensitivity of 1H,1H nuclear Overhauser effects. J Biomol NMR. 2022 Dec 19. doi: 10.1007/s10858-022-00407-y. Epub ahead of print. PMID: 36534224.

Manu et al. Design and applications of water irradiation devoid RF pulses for ultra-high field biomolecular NMR spectroscopy. Phys Chem Chem Phys. 2022 Aug 10;24(31):18477-18481. doi: 10.1039/d2cp01744j. PMID: 35895081; PMCID: PMC9578148.

Gopinath et al. Multi-receiver solid-state NMR using polarization optimized experiments (POE) at ultrafast magic angle spinning. J Biomol NMR. 2020 May;74(4-5):267-285. doi: 10.1007/s10858-020-00316-y. Epub 2020 Apr 24. PMID: 32333193; PMCID: PMC7236978.

Gopinath and Veglia Experimental Aspects of Polarization Optimized Experiments (POE) for Magic Angle Spinning Solid-State NMR of Microcrystalline and Membrane-Bound Proteins. Methods Mol Biol. 2018;1688:37-53. doi: 10.1007/978-1-4939-7386-6_2. PMID: 29151203.