Publications by authors named "Gianluigi Veglia"

180 Publications

Raf Kinase Inhibitory Protein regulates the cAMP-dependent protein kinase signaling pathway through a positive feedback loop.

Proc Natl Acad Sci U S A 2022 Jun 13;119(25):e2121867119. Epub 2022 Jun 13.

Ben May Department for Cancer Research, University of Chicago, Chicago, IL 60637.

Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the β-adrenergic receptor (β-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes β-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the β-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.
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http://dx.doi.org/10.1073/pnas.2121867119DOI Listing
June 2022

PHRONESIS: A One-Shot Approach for Sequential Assignment of Protein Resonances by Ultrafast MAS Solid-State NMR Spectroscopy.

Chemphyschem 2022 May 2:e202200127. Epub 2022 May 2.

Department of Biochemistry, Molecular Biology & Biophysics -, University of Minnesota, 321 Church St. SE, Minneapolis, MN 55455, USA.

Solid-state NMR (ssNMR) spectroscopy has emerged as the method of choice to analyze the structural dynamics of fibrillar, membrane-bound, and crystalline proteins that are recalcitrant to other structural techniques. Recently, H detection under fast magic angle spinning and multiple acquisition ssNMR techniques have propelled the structural analysis of complex biomacromolecules. However, data acquisition and resonance-specific assignments remain a bottleneck for this technique. Here, we present a comprehensive multi-acquisition experiment (PHRONESIS) that simultaneously generates up to ten 3D H-detected ssNMR spectra. PHRONESIS utilizes broadband transfer and selective pulses to drive multiple independent polarization pathways. High selectivity excitation and de-excitation of specific resonances were achieved by high-fidelity selective pulses that were designed using a combination of an evolutionary algorithm and artificial intelligence. We demonstrated the power of this approach with microcrystalline U- C, N GB1 protein, reaching 100 % of the resonance assignments using one data set of ten 3D experiments. The strategy outlined in this work opens up new avenues for implementing novel H-detected multi-acquisition ssNMR experiments to speed up and expand the application to larger biomolecular systems.
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http://dx.doi.org/10.1002/cphc.202200127DOI Listing
May 2022

A kink in DWORF helical structure controls the activation of the sarcoplasmic reticulum Ca-ATPase.

Structure 2022 03 6;30(3):360-370.e6. Epub 2021 Dec 6.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, Minneapolis, MN 55455, USA; Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

SERCA is a P-type ATPase embedded in the sarcoplasmic reticulum and plays a central role in muscle relaxation. SERCA's function is regulated by single-pass membrane proteins called regulins. Unlike other regulins, dwarf open reading frame (DWORF) expressed in cardiac muscle has a unique activating effect. Here, we determine the structure and topology of DWORF in lipid bilayers using a combination of oriented sample solid-state NMR spectroscopy and replica-averaged orientationally restrained molecular dynamics. We found that DWORF's structural topology consists of a dynamic N-terminal domain, an amphipathic juxtamembrane helix that crosses the lipid groups at an angle of 64°, and a transmembrane C-terminal helix with an angle of 32°. A kink induced by Pro15, unique to DWORF, separates the two helical domains. A single Pro15Ala mutant significantly decreases the kink and eliminates DWORF's activating effect on SERCA. Overall, our findings directly link DWORF's structural topology to its activating effect on SERCA.
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http://dx.doi.org/10.1016/j.str.2021.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8897251PMC
March 2022

Structural basis for sarcolipin's regulation of muscle thermogenesis by the sarcoplasmic reticulum Ca-ATPase.

Sci Adv 2021 Nov 26;7(48):eabi7154. Epub 2021 Nov 26.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

The sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) plays a central role in muscle contractility and nonshivering thermogenesis. SERCA is regulated by sarcolipin (SLN), a single-pass membrane protein that uncouples Ca transport from ATP hydrolysis, promoting futile enzymatic cycles and heat generation. The molecular determinants for regulating heat release by the SERCA/SLN complex are unclear. Using thermocalorimetry, chemical cross-linking, and solid-state NMR spectroscopy in oriented phospholipid bicelles, we show that SERCA’s functional uncoupling and heat release rate are dictated by specific SERCA/SLN intramembrane interactions, with the carboxyl-terminal residues anchoring SLN to the SR membrane in an inhibitory topology. Systematic deletion of the carboxyl terminus does not prevent the SERCA/SLN complex formation but reduces uncoupling in a graded manner. These studies emphasize the critical role of lipids in defining the active topology of SLN and modulating the heat release rate by the SERCA/SLN complex, with implications in fat metabolism and basal metabolic rate.
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http://dx.doi.org/10.1126/sciadv.abi7154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8626070PMC
November 2021

Is Disrupted Nucleotide-Substrate Cooperativity a Common Trait for Cushing's Syndrome Driving Mutations of Protein Kinase A?

J Mol Biol 2021 09 3;433(18):167123. Epub 2021 Jul 3.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA; Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

Somatic mutations in the PRKACA gene encoding the catalytic α subunit of protein kinase A (PKA-C) are responsible for cortisol-producing adrenocortical adenomas. These benign neoplasms contribute to the development of Cushing's syndrome. The majority of these mutations occur at the interface between the two lobes of PKA-C and interfere with the enzyme's ability to recognize substrates and regulatory (R) subunits, leading to aberrant phosphorylation patterns and activation. Rarely, patients with similar phenotypes carry an allosteric mutation, E31V, located at the C-terminal end of the αA-helix and adjacent to the αC-helix, but structurally distinct from the PKA-C/R subunit interface mutations. Using a combination of solution NMR, thermodynamics, kinetic assays, and molecular dynamics simulations, we show that the E31V allosteric mutation disrupts central communication nodes between the N- and C- lobes of the enzyme as well as nucleotide-substrate binding cooperativity, a hallmark for kinases' substrate fidelity and regulation. For both orthosteric (L205R and W196R) and allosteric (E31V) Cushing's syndrome mutants, the loss of binding cooperativity is proportional to the density of the intramolecular allosteric network. This structure-activity relationship suggests a possible common mechanism for Cushing's syndrome driving mutations in which decreased nucleotide/substrate binding cooperativity is linked to loss in substrate fidelity and dysfunctional regulation.
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http://dx.doi.org/10.1016/j.jmb.2021.167123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409435PMC
September 2021

Activation mechanism of cryptochrome through an allosteric switch.

Sci Adv 2021 Jun 18;7(25). Epub 2021 Jun 18.

Department of Chemistry and Supercomputing Institute, University of Minnesota, Minneapolis, MN 55455, USA.

Cryptochromes are signaling proteins activated by photoexcitation of the flavin adenine dinucleotide (FAD) cofactor. Although extensive research has been performed, the mechanism for this allosteric process is still unknown. We constructed three computational models, corresponding to different redox states of the FAD cofactor in cryptochrome (dCRY). Analyses of the dynamics trajectories reveal that the activation process occurs in the semiquinone state FAD, resulting from excited-state electron transfer. The Arg-Asp salt bridge acts as an allosteric switch, regulated by the change in the redox state of FAD. In turn, Asp forms new hydrogen bonds, connecting allosteric networks of the amino-terminal and carboxyl-terminal domains initially separated in the resting state. The expansion to a global dynamic network leads to enhanced protein fluctuations, an increase in the radius of gyration, and the expulsion of the carboxyl-terminal tail. These structural features are in accord with mutations and spectroscopic experiments.
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http://dx.doi.org/10.1126/sciadv.abg3815DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213227PMC
June 2021

Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca and phosphorylation.

Elife 2021 05 12;10. Epub 2021 May 12.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, United States.

Phospholamban (PLN) is a mini-membrane protein that directly controls the cardiac Ca-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 Ca-ATPase (SERCA), keeping this enzyme's function within a narrow physiological window. PLN phosphorylation by cAMP-dependent protein kinase A or increase in Ca 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 Ca 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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http://dx.doi.org/10.7554/eLife.66226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184213PMC
May 2021

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.

Commun Biol 2021 03 10;4(1):321. Epub 2021 Mar 10.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-C) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-C has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-C alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.
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http://dx.doi.org/10.1038/s42003-021-01819-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7946884PMC
March 2021

Solid-State NMR of Membrane Proteins in Lipid Bilayers: To Spin or Not To Spin?

Acc Chem Res 2021 03 3;54(6):1430-1439. Epub 2021 Mar 3.

Membrane proteins mediate a plethora of cellular functions and represent important targets for drug development. Unlike soluble proteins, membrane proteins require native-like environments to fold correctly and be active. Therefore, modern structural biology techniques have aimed to determine the structure and dynamics of these membrane proteins at physiological temperature and in liquid crystalline lipid bilayers. With the flourishing of new NMR methodologies and improvements in sample preparations, magic angle spinning (MAS) and oriented sample solid-state NMR (OS-ssNMR) spectroscopy of membrane proteins is experiencing a new renaissance. Born as antagonistic approaches, these techniques nowadays offer complementary information on the structural topology and dynamics of membrane proteins reconstituted in lipid membranes. By spinning biosolid samples at the magic angle (θ = 54.7°), MAS NMR experiments remove the intrinsic anisotropy of the NMR interactions, increasing spectral resolution. Internuclear spin interactions (spin exchange) are reintroduced by RF pulses, providing distances and torsion angles to determine secondary, tertiary, and quaternary structures of membrane proteins. OS-ssNMR, on the other hand, directly detects anisotropic NMR parameters such as dipolar couplings (DC) and anisotropic chemical shifts (CS), providing orientational constraints to determine the architecture (i.e., topology) of membrane proteins relative to the lipid membrane. Defining the orientation of membrane proteins and their interactions with lipid membranes is of paramount importance since lipid-protein interactions can shape membrane protein conformations and ultimately define their functional states.In this Account, we report selected studies from our group integrating MAS and OS-ssNMR techniques to give a comprehensive view of the biological processes occurring at cellular membranes. We focus on the main experiments for both techniques, with an emphasis on new implementation to increase both sensitivity and spectral resolution. We also describe how the structural constraints derived from both isotropic and anisotropic NMR parameters are integrated into dynamic structural modeling using replica-averaged orientational-restrained molecular dynamics simulations (RAOR-MD). We showcase small membrane proteins that are involved in Ca transport and regulate cardiac and skeletal muscle contractility: phospholamban (PLN, 6 kDa), sarcolipin (SLN, 4 kDa), and DWORF (4 kDa). We summarize our results for the structures of these polypeptides free and in complex with the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA, 110 kDa). Additionally, we illustrate the progress toward the determination of the structural topology of a six transmembrane protein associated with succinate and acetate transport (SatP, hexamer 120 kDa). From these examples, the integrated MAS and OS-ssNMR approach, in combination with modern computational methods, emerges as a way to overcome the challenges posed by studying large membrane protein systems.
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http://dx.doi.org/10.1021/acs.accounts.0c00670DOI Listing
March 2021

Allostery governs Cdk2 activation and differential recognition of CDK inhibitors.

Nat Chem Biol 2021 04 1;17(4):456-464. Epub 2021 Feb 1.

Department of Pharmacology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.

Cyclin-dependent kinases (CDKs) are the master regulators of the eukaryotic cell cycle. To become activated, CDKs require both regulatory phosphorylation and binding of a cognate cyclin subunit. We studied the activation process of the G1/S kinase Cdk2 in solution and developed a thermodynamic model that describes the allosteric coupling between regulatory phosphorylation, cyclin binding and inhibitor binding. The results explain why monomeric Cdk2 lacks activity despite sampling an active-like state, reveal that regulatory phosphorylation enhances allosteric coupling with the cyclin subunit and show that this coupling underlies differential recognition of Cdk2 and Cdk4 inhibitors. We identify an allosteric hub that has diverged between Cdk2 and Cdk4 and show that this hub controls the strength of allosteric coupling. The altered allosteric wiring of Cdk4 leads to compromised activity toward generic peptide substrates and comparative specialization toward its primary substrate retinoblastoma (RB).
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http://dx.doi.org/10.1038/s41589-020-00725-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990704PMC
April 2021

Met125 is essential for maintaining the structural integrity of calmodulin's C-terminal domain.

Sci Rep 2020 12 7;10(1):21320. Epub 2020 Dec 7.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 321 Church Street SE, Minneapolis, MN, 55455, USA.

We have used NMR and circular dichroism spectroscopy to investigate the structural and dynamic effects of oxidation on calmodulin (CaM), using peroxide and the Met to Gln oximimetic mutations. CaM is a Ca-sensitive regulatory protein that interacts with numerous targets. Due to its high methionine content, CaM is highly susceptible to oxidation by reactive oxygen species under conditions of cell stress and age-related muscle degeneration. CaM oxidation alters regulation of a host of CaM's protein targets, emphasizing the importance of understanding the mechanism of CaM oxidation in muscle degeneration and overall physiology. It has been shown that the M125Q CaM mutant can mimic the functional effects of methionine oxidation on CaM's regulation of the calcium release channel, ryanodine receptor (RyR). We report here that the M125Q mutation causes a localized unfolding of the C-terminal lobe of CaM, preventing the formation of a hydrophobic cluster of residues near the EF-hand Ca binding sites. NMR analysis of CaM oxidation by peroxide offers further insights into the susceptibility of CaM's Met residues to oxidation and the resulting structural effects. These results further resolve oxidation-driven structural perturbation of CaM, with implications for RyR regulation and the decay of muscle function in aging.
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http://dx.doi.org/10.1038/s41598-020-78270-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721703PMC
December 2020

Dynamical and allosteric regulation of photoprotection in light harvesting complex II.

Sci China Chem 2020 Aug 15;63(8):1121-1133. Epub 2020 Jun 15.

Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing 100190, China.

Major light-harvesting complex of photosystem II (LHCII) plays a dual role in light-harvesting and excited energy dissipation to protect photodamage from excess energy. The regulatory switch is induced by increased acidity, temperature or both. However, the molecular origin of the protein dynamics at the atomic level is still unknown. We carried out temperature-jump time-resolved infrared spectroscopy and molecular dynamics simulations to determine the energy quenching dynamics and conformational changes of LHCII trimers. We found that the spontaneous formation of a pair of local α-helices from the 3-helix E/loop and the C-terminal coil of the neighboring monomer, in response to the increased environmental temperature and/or acidity, induces a scissoring motion of transmembrane helices A and B, shifting the conformational equilibrium to a more open state, with an increased angle between the associated carotenoids. The dynamical allosteric conformation change leads to close contacts between the first excited state of carotenoid lutein 1 and chlorophyll pigments, facilitating the fluorescence quenching. Based on these results, we suggest a unified mechanism by which the LHCII trimer controls the dissipation of excess excited energy in response to increased temperature and acidity, as an intrinsic result of intense sun light in plant photosynthesis.
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http://dx.doi.org/10.1007/s11426-020-9771-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643867PMC
August 2020

A theoretical assessment of structure determination of multi-span membrane proteins by oriented sample solid-state NMR spectroscopy.

Aust J Chem 2020 20;73(3):246-251. Epub 2019 Sep 20.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

Oriented sample solid state NMR (OS-ssNMR) spectroscopy allows direct determination of the structure and topology of membrane proteins reconstituted into aligned lipid bilayers. While OS-ssNMR theoretically has no upper size limit, its application to multi-span membrane proteins has not been established since most studies have been restricted to single or dual span proteins and peptides. Here, we present a critical assessment of the application of this method to multi-span membrane proteins. We used molecular dynamics simulations to back-calculate [N-H] separated local field (SLF) spectra from a G protein-coupled receptor (GPCR) and show that fully resolved spectra can be obtained theoretically for a multi-span membrane protein with currently achievable resonance linewidths.
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http://dx.doi.org/10.1071/CH19307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643874PMC
September 2019

CHESPA/CHESCA-SPARKY: automated NMR data analysis plugins for SPARKY to map protein allostery.

Bioinformatics 2021 05;37(8):1176-1177

Biochemistry Department, National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, Madison, WI 53717, USA.

Motivation: Correlated Nuclear Magnetic Resonance (NMR) chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other NMR analysis software packages, we introduce here integrated plugins for NMRFAM-SPARKY that implement the seamless detection and visualization of allosteric networks.

Availability And Implementation: CHESCA-SPARKY and CHESPA-SPARKY are available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). The assigned spectra involved in this study and tutorial videos using this dataset are available at https://sites.google.com/view/chescachespa-sparky.

Supplementary Information: Supplementary data are available at Bioinformatics Online.
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http://dx.doi.org/10.1093/bioinformatics/btaa781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150123PMC
May 2021

Molecular Mechanism for the Suppression of Alpha Synuclein Membrane Toxicity by an Unconventional Extracellular Chaperone.

J Am Chem Soc 2020 05 8;142(21):9686-9699. Epub 2020 May 8.

Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton ON L8S 4M1, Canada.

Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αS) toxicity through a three-pronged mechanism. First, endogenous HSA targets αS with sub-μM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.
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http://dx.doi.org/10.1021/jacs.0c01894DOI Listing
May 2020

Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

Elife 2020 04 27;9. Epub 2020 Apr 27.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, United States.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.
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http://dx.doi.org/10.7554/eLife.55607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7234811PMC
April 2020

Multi-receiver solid-state NMR using polarization optimized experiments (POE) at ultrafast magic angle spinning.

J Biomol NMR 2020 May 24;74(4-5):267-285. Epub 2020 Apr 24.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, Minneapolis, MN, 55455, USA.

Ultrafast magic angle spinning (MAS) technology and H detection have dramatically enhanced the sensitivity of solid-state NMR (ssNMR) spectroscopy of biopolymers. We previously showed that, when combined with polarization optimized experiments (POE), these advancements enable the simultaneous acquisition of multi-dimensional H- or C-detected experiments using a single receiver. Here, we propose a new sub-class within the POE family, namely HC-DUMAS, HC-MEIOSIS, and HC-MAeSTOSO, that utilize dual receiver technology for the simultaneous detection of H and C nuclei. We also expand this approach to record H-, C-, and N-detected homonuclear 2D spectra simultaneously using three independent receivers. The combination of POE and multi-receiver technology will further shorten the total experimental time of ssNMR experiments for biological solids.
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http://dx.doi.org/10.1007/s10858-020-00316-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236978PMC
May 2020

Protein kinase A in the neutron beam: Insights for catalysis from directly observing protons.

Methods Enzymol 2020 17;634:311-331. Epub 2020 Jan 17.

Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States. Electronic address:

Protein kinases transmit chemical signals by phosphorylating substrate proteins, thus regulating a multitude of cellular processes. cAMP-dependent protein kinase (PKA), a prototypical enzyme for the whole kinase family, has been the focus of research for several decades, however, the details of the chemical mechanism of phosphoryl group transfer have remained unknown. We used neutron crystallography to map key proton sites and hydrogen bonding interactions in the PKA catalytic subunit (PKAc) in a product complex containing adenosine diphosphate (ADP) and the phosphorylated high affinity protein kinase substrate (pPKS) peptide. To improve neutron diffraction, we deuterated PKAc allowing us to use very small crystals. In the product complex, the phosphoryl group of pPKS is protonated whereas the catalytic Asp166 is not. H/D exchange analysis of the main-chain amides and comparison with the NMR analysis of PKAc with inhibitor peptide complex revealed exchangeable amides that may distinguish the catalytic and inhibited states.
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http://dx.doi.org/10.1016/bs.mie.2019.12.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494206PMC
June 2021

Nanosecond-Timescale Dynamics and Conformational Heterogeneity in Human GCK Regulation and Disease.

Biophys J 2020 03 14;118(5):1109-1118. Epub 2020 Jan 14.

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida. Electronic address:

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.
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http://dx.doi.org/10.1016/j.bpj.2019.12.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7063420PMC
March 2020

PISA-SPARKY: an interactive SPARKY plugin to analyze oriented solid-state NMR spectra of helical membrane proteins.

Bioinformatics 2020 05;36(9):2915-2916

National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, WI 53706, USA.

Motivation: Two-dimensional [15N-1H] separated local field solid-state nuclear magnetic resonance (NMR) experiments of membrane proteins aligned in lipid bilayers provide tilt and rotation angles for α-helical segments using Polar Index Slant Angle (PISA)-wheel models. No integrated software has been made available for data analysis and visualization.

Results: We have developed the PISA-SPARKY plugin to seamlessly integrate PISA-wheel modeling into the NMRFAM-SPARKY platform. The plugin performs basic simulations, exhaustive fitting against experimental spectra, error analysis and dipolar and chemical shift wave plotting. The plugin also supports PyMOL integration and handling of parameters that describe variable alignment and dynamic scaling encountered with magnetically aligned media, ensuring optimal fitting and generation of restraints for structure calculation.

Availability And Implementation: PISA-SPARKY is freely available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers of the SBGrid (https://sbgrid.org). The pisa.py script is available and documented on GitHub (https://github.com/weberdak/pisa.py) along with a tutorial video and sample data.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203746PMC
May 2020

Proton-detected polarization optimized experiments (POE) using ultrafast magic angle spinning solid-state NMR: Multi-acquisition of membrane protein spectra.

J Magn Reson 2020 01 28;310:106664. Epub 2019 Nov 28.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, United States; Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, United States. Electronic address:

Proton-detected solid-state NMR (ssNMR) spectroscopy has dramatically improved the sensitivity and resolution of fast magic angle spinning (MAS) methods. While relatively straightforward for fibers and crystalline samples, the routine application of these techniques to membrane protein samples is still challenging. This is due to the low sensitivity of these samples, which require high lipid:protein ratios to maintain the structural and functional integrity of membrane proteins. We previously introduced a family of novel polarization optimized experiments (POE) that enable to make the best of nuclear polarization and obtain multiple-acquisitions from a single pulse sequence and one receiver. Here, we present the H-detected versions of POE using ultrafast MAS ssNMR. Specifically, we implemented proton detection into our three main POE strategies, H-DUMAS, H-MEIOSIS, and H-MAeSTOSO, achieving the acquisition of up to ten different experiments using a single pulse sequence. We tested these experiments on a model compound N-Acetyl-Val-Leu dipeptide and applied to a six transmembrane acetate transporter, SatP, reconstituted in lipid membranes. These new methods will speed up the spectroscopy of challenging biomacromolecules such as membrane proteins.
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http://dx.doi.org/10.1016/j.jmr.2019.106664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003683PMC
January 2020

Cushing's syndrome driver mutation disrupts protein kinase A allosteric network, altering both regulation and substrate specificity.

Sci Adv 2019 08 28;5(8):eaaw9298. Epub 2019 Aug 28.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.

Genetic alterations in the gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing's syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-C is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.
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http://dx.doi.org/10.1126/sciadv.aaw9298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713507PMC
August 2019

Improving the quality of oriented membrane protein spectra using heat-compensated separated local field experiments.

J Biomol NMR 2019 Nov 28;73(10-11):617-624. Epub 2019 Aug 28.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA.

Oriented sample solid-state NMR (OS-ssNMR) spectroscopy is a powerful technique to determine the topology of membrane proteins in oriented lipid bilayers. Separated local field (SLF) experiments are central to this technique as they provide first-order orientational restraints, i.e., dipolar couplings and anisotropic chemical shifts. Despite the use of low-E (or E-free) probes, the heat generated during the execution of 2D and 3D SLF pulse sequences causes sizeable line-shape distortions. Here, we propose a new heat-compensated SE-SAMPI4 (hcSE-SAMPI4) pulse sequence that holds the temperature constant for the duration of the experiment. This modification of the SE-SAMPI4 results in sharper and more intense resonances without line-shape distortions. The spectral improvements are even more apparent when paramagnetic relaxation agents are used to speed up data collection. We tested the hcSE-SAMPI4 pulse sequence on a single-span membrane protein, sarcolipin (SLN), reconstituted in magnetically aligned lipid bicelles. In addition to eliminating peak distortions, the hcSE-SAMPI4 experiment increased the average signal-to-noise ratio by 20% with respect to the original SE-SAMPI4.
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http://dx.doi.org/10.1007/s10858-019-00273-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861693PMC
November 2019

Intrinsically disordered HAX-1 regulates Ca cycling by interacting with lipid membranes and the phospholamban cytoplasmic region.

Biochim Biophys Acta Biomembr 2020 01 7;1862(1):183034. Epub 2019 Aug 7.

Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA; Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address:

Hematopoietic-substrate-1 associated protein X-1 (HAX-1) is a 279 amino acid protein expressed ubiquitously. In cardiac muscle, HAX-1 was found to modulate the sarcoendoplasmic reticulum calcium ATPase (SERCA) by shifting its apparent Ca affinity (pCa). It has been hypothesized that HAX-1 binds phospholamban (PLN), enhancing its inhibitory function on SERCA. HAX-1 effects are reversed by cAMP-dependent protein kinase A that phosphorylates PLN at Ser16. To date, the molecular mechanisms for HAX-1 regulation of the SERCA/PLN complex are still unknown. Using enzymatic, in cell assays, circular dichroism, and NMR spectroscopy, we found that in the absence of a binding partner HAX-1 is essentially disordered and adopts a partial secondary structure upon interaction with lipid membranes. Also, HAX-1 interacts with the cytoplasmic region of monomeric and pentameric PLN as detected by NMR and in cell FRET assays, respectively. We propose that the regulation of the SERCA/PLN complex by HAX-1 is mediated by its interactions with lipid membranes, adding another layer of control in Ca homeostatic balance in the heart muscle.
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http://dx.doi.org/10.1016/j.bbamem.2019.183034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899184PMC
January 2020

Use of paramagnetic systems to speed-up NMR data acquisition and for structural and dynamic studies.

Solid State Nucl Magn Reson 2019 10 12;102:36-46. Epub 2019 Jul 12.

Department of Chemistry, University of Michigan, Ann Arbor, MI, USA; Biophysics, Biomedical Engineering, Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, USA. Electronic address:

NMR spectroscopy is a powerful experimental technique to study biological systems at the atomic resolution. However, its intrinsic low sensitivity results in long acquisition times that in extreme cases lasts for days (or even weeks) often exceeding the lifetime of the sample under investigation. Different paramagnetic agents have been used in an effort to decrease the spin-lattice (T) relaxation times of the studied nuclei, which are the main cause for long acquisition times necessary for signal averaging to enhance the signal-to-noise ratio of NMR spectra. Consequently, most of the experimental time is "wasted" in waiting for the magnetization to recover between successive scans. In this review, we discuss how to set up an optimal paramagnetic relaxation enhancement (PRE) system to effectively reduce the T relaxation times avoiding significant broadening of NMR signals. Additionally, we describe how PRE-agents can be used to provide structural and dynamic information and can even be used to follow the intermediates of chemical reactions and to speed-up data acquisition. We also describe the unique challenges and benefits associated with the application of PRE to solid-state NMR spectroscopy, explaining how the use of PREs is more complex for membrane mimetic systems as PREs can also be exploited to change the alignment of oriented membrane systems. Functionalization of membrane mimetics, such as bicelles, can provide a controlled region of paramagnetic effect that has the potential, together with the desired alignment, to provide crucial biologically relevant structural information. And finally, we discuss how paramagnetic metals can be utilized to further increase the dynamic nuclear polarization (DNP) effects and how to preserve the enhancements when dissolution DNP is implemented.
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http://dx.doi.org/10.1016/j.ssnmr.2019.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698407PMC
October 2019

Dynamic allostery-based molecular workings of kinase:peptide complexes.

Proc Natl Acad Sci U S A 2019 07 8;116(30):15052-15061. Epub 2019 Jul 8.

Department of Pharmacology, University of California San Diego, La Jolla, CA 92093;

A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.
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http://dx.doi.org/10.1073/pnas.1900163116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660753PMC
July 2019

T* weighted Deconvolution of NMR Spectra: Application to 2D Homonuclear MAS Solid-State NMR of Membrane Proteins.

Sci Rep 2019 06 3;9(1):8225. Epub 2019 Jun 3.

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.

2D homonuclear NMR spectroscopy is an essential technique to characterize small and large molecules, such as organic compounds, metabolites, and biomacromolecules at atomic resolution. However, for complex samples 2D homonuclear spectra display poor resolution, making spectral assignment very cumbersome. Here, we propose a new method that exploits the differential T* relaxation times of individual resonances and resolves the 2D NMR peaks into pseudo-3D spectra, where time is the 3 dimension. T* weIghted DEconvolution or TIDE analyzes individual free induction decays (FIDs) and dissects them into sub-FIDs that are transformed into pseudo-3D spectra combining Fourier transformation and covariance NMR. TIDE achieves higher resolution and sensitivity for NMR spectra than classical covariance NMR reducing offset-dependent artifacts. We demonstrate the performance of TIDE for magic angle spinning (MAS) [C,C]-DARR NMR spectra of single- and multi-span membrane proteins embedded in lipid bilayers. Since TIDE is applicable to all type of homonuclear correlation experiments for liquid and solid samples, we anticipate that it will be a general method for processing NMR data of biomacromolecules, complex mixtures of metabolites as well as material samples.
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http://dx.doi.org/10.1038/s41598-019-44461-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546711PMC
June 2019

Cysteine-ethylation of tissue-extracted membrane proteins as a tool to detect conformational states by solid-state NMR spectroscopy.

Methods Enzymol 2019 8;621:281-304. Epub 2019 Mar 8.

Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, United States; Department of Chemistry, University of Minnesota, Minneapolis, MN, United States. Electronic address:

Solid-state NMR (ssNMR) is an ideal tool to study structure and dynamics of membrane proteins in their native lipid environment. In principle, ssNMR has no size limitations. However, this feature is rarely exploited as large membrane proteins display severe resonance overlap. In addition, dismal yields from recombinant bacterial expression systems limit severely spectroscopic characterization of membrane proteins. For very large mammalian membrane proteins, extraction from the original organism remains the most viable approach. In this case, NMR-observable nuclei must be introduced post-translationally, but the approaches developed so far are rather scarce. Here, we detail the synthesis and engineering of a reactive C-ethylmethanethiosulfonate (C-EMTS) reagent for the post-translational alkylation of cysteine sidechains of a 110kDa sarcoplasmic reticulum Ca-ATPase (SERCA) extracted from rabbit skeletal muscle tissue. When reconstituted into liposomes, it is possible to resolve the resonances of the engineered ethyl groups by magic-angle spinning (MAS) 2D [C,C]-DARR experiments. Notably, the ethyl-group modification does not perturb the function of SERCA, yielding well-resolved C-C fingerprints that are used to image its structural states in the catalytic cycle and filtering out overwhelming naturally-abundant C nuclei signals arising from the enzyme and lipids. We anticipate that this approach will be used together with F NMR to monitor conformational transitions of enzymes and proteins that are difficult to express recombinantly.
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http://dx.doi.org/10.1016/bs.mie.2019.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418180PMC
February 2020

More than a Liquid Junction: Effect of Stirring, Flow Rate, and Inward and Outward Electrolyte Diffusion on Reference Electrodes with Salt Bridges Contained in Nanoporous Glass.

Anal Chem 2019 Jun 7;91(12):7698-7704. Epub 2019 Jun 7.

The Henderson equation is usually used to calculate liquid-junction potentials between miscible electrolyte solutions. However, the potentials of reference electrodes that comprise an electrolyte-filled nanoporous glass frit may also be affected by charge screening. As reported previously, when the Debye length approaches or surpasses the glass pore diameter, reference potentials depend on the composition of the bridge electrolyte, the pore size of the frit, and the concentration of electrolyte in the sample. We report here that stirring of samples may alter the reference potential as it affects the electrolyte concentration in the section of the nanoporous glass frit that is facing the sample solution. When the flow rate of bridge electrolyte into the sample is small, convective mass transport of sample into the nanoporous frit occurs. The depth of penetration into the frit is only a few nanometers but, despite the use of concentrated salt bridges, this is enough to affect the extent of electrostatic screening when samples of low ionic strength are measured. Mixing of sample and salt bridge solutions-and in particular penetration of sample components into the frit-was optically monitored by observation of a deeply colored Fe[(SCN)(HO)] complex that formed in situ exclusively in the region where the sample and salt bridge mixed. Importantly, because flow through nanoporous frits is very slow, mass transport through these frits is dominated by diffusion. Consequently, over as little as 1 h, reference electrode frits with low flow rates become contaminated with sample components and undergo depletion of electrolyte within the frit to a depth of several millimeters, which can negatively affect subsequent experiments.
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http://dx.doi.org/10.1021/acs.analchem.9b00876DOI Listing
June 2019
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