14 research outputs found
Structure and Topology of the 114kDa Serca-Sarcolipin Complex by Solid State NMR Spectroscopy
The TLQP-21 Peptide Activates the G-Protein-Coupled Receptor C3aR1 via a Folding-upon-Binding Mechanism
TLQP-21, a VGF-encoded peptide is emerging as a novel target for obesity-associated disorders. TLQP-21 is found in the sympathetic nerve terminals in the adipose tissue and targets the G-protein-coupled-receptor (GPCR) Complement-3a-Receptor1 (C3aR1). So far, the mechanisms of TLQP-21-induced receptor activation remained unexplored. Here, we report that TLQP-21 is intrinsically disordered and undergoes a disorder-to-order transition, adopting an α-helical conformation, upon targeting cells expressing the C3aR1. We determined that the hot spots for TLQP-21 are located at the C-terminus, with mutations in the last four amino acids progressively reducing the bioactivity and, a single site mutation (R21A) or C-terminal amidation abolishing its function completely. Interestingly, the human TLQP-21 sequence carrying a S20A substitution activates the human C3aR1 receptor with lower potency compared to the rodent sequence. These studies reveal the mechanism of action of TLQP-21 and provide molecular templates for designing agonists and antagonists to modulate C3aR1 functions
Characterization of Calmodulin Binding to the Ryanodine Receptor by Solution and Solid-State NMR
Role of the Strength of Drug–Polymer Interactions on the Molecular Mobility and Crystallization Inhibition in Ketoconazole Solid Dispersions
The effects of specific drug–polymer
interactions (ionic
or hydrogen-bonding) on the molecular mobility of model amorphous
solid dispersions (ASDs) were investigated. ASDs of ketoconazole (KTZ),
a weakly basic drug, with each of polyÂ(acrylic acid) (PAA), polyÂ(2-hydroxyethyl
methacrylate) (PHEMA), and polyvinylpyrrolidone (PVP) were prepared.
Drug–polymer interactions in the ASDs were evaluated by infrared
and solid-state NMR, the molecular mobility quantified by dielectric
spectroscopy, and crystallization onset monitored by differential
scanning calorimetry (DSC) and variable temperature X-ray diffractometry
(VTXRD). KTZ likely exhibited ionic interactions with PAA, hydrogen-bonding
with PHEMA, and weaker dipole–dipole interactions with PVP.
On the basis of dielectric spectroscopy, the α-relaxation times
of the ASDs followed the order: PAA > PHEMA > PVP. In addition,
the
presence of ionic interactions also translated to a dramatic and disproportionate
decrease in mobility as a function of polymer concentration. On the
basis of both DSC and VTXRD, an increase in strength of interaction
translated to higher crystallization onset temperature and a decrease
in extent of crystallization. Stronger drug–polymer interactions,
by reducing the molecular mobility, can potentially delay the crystallization
onset temperature as well as crystallization extent
Replica-averaged orientational-restrained ensembles of DWORF and P15A-DWORF in lipid bilayers
Replica-averaged orientational-restrained molecular dynamics (RAOR-MD) ensembles of DWORF and the DWORF-P15A mutant in lipid bilayers. Restrained to 15N chemical shift anisotropy and 1H-15N dipolar coupling data obtained from oriented sample solid-state NMR spectroscopy.National Institutes of Health (R01HL143816, R01HL092321)American Heart Association (19POST34420009
Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and 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
Ca<sup>2+</sup> ATPase Conformational Transitions in Lipid Bilayers Mapped by Site-directed Ethylation and Solid-State NMR
To transmit signals across cellular
compartments, many membrane-embedded
enzymes undergo extensive conformational rearrangements. Monitoring
these events in lipid bilayers by NMR at atomic resolution has been
challenging due to the large size of these systems. It is further
exacerbated for large mammalian proteins that are difficult to express
and label with NMR-active isotopes. Here, we synthesized and engineered <sup>13</sup>C ethyl groups on native cysteines to map the structural
transitions of the sarcoplasmic reticulum Ca<sup>2+</sup>-ATPase,
a 110 kDa transmembrane enzyme that transports Ca<sup>2+</sup> into
the sarcoplasmic reticulum. Using magic angle spinning NMR, we monitored
the chemical shifts of the methylene and methyl groups of the derivatized
cysteine residues along the major steps of the enzymatic cycle. The
methylene chemical shifts are sensitive to the ATPase conformational
changes induced upon nucleotide and Ca<sup>2+</sup> ion binding and
are ideal probes for active and inactive states of the enzyme. This
new approach is extendable to large mammalian enzymes and signaling
proteins with native or engineered cysteine residues in their amino
acid sequence
Author response: Structural basis for allosteric control of the SERCA-Phospholamban membrane complex by Ca2+ and phosphorylation
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Ensembles from dynamic refinement of non-phosphorylated and phosphorylated phospholamban-SERCA complexes
Full GROMACS ensembles of SERCA-PLN and SERCA-pS16PLN complexes (40 ps time-step, 10200 frames, 400 ns)
- SERCA_PLN_E2_apo_Ensemble (6.6 GB). Topology file: SERCA_PLN_E2_apo_frame0.gro (7.8 MB)
- SERCA_PLN_E2_pS16_Ensemble.xtc (6.6 GB). Topology file: SERCA_PLN_E2_pS16_frame0.gro (7.8 MB)
Stripped GROMACS ensembles of SERCA-PLN and SERCA-pS16PLN complexes (40 ps time-step, 10200 frames, 400 ns) - no water or lipids
- nonP_PLN_SERCA_E2_8rep_dt40.xtc (633.8 MB). Topology file: nonP_PLN_SERCA_E2.gro (735.9 kB)
- pS16_PLN_SERCA_E2_8rep_dt40.xtc (634.1 MB). Topology file: pS16_PLN_SERCA_E2.gro (736.0 kB)
Configuration and restraint files use to generate ensembles
Analytical scripts and processed data for:
- Principal component analysis
- Distance measurements
- Contact analysis
- Helix allostery
Raw experimental data:
- All SE-SAMPI4 spectra of PLN and pPLN alone and in complex with SERCA
- Raw FID files
- Processing script (NMRPipe)
- AssignmentsAnalysis, setup and ensembles of the dynamic refinement of phospholamban-SERCA complexes in the calcium-free E2 state. Includes trajectory files, analytic scripts, restraint files, setup files, simulation code, raw data (including OS-ssNMR data) and processed data used for the associated citations as per transparent reporting requirements.National Institutes of Health R01GM064742National Institutes of Health R01HL144100National Institutes of Health R01HL139065National Institutes of Health R37AG026160American Heart Association Postdoctoral Fellowship 19POST34420009European Research Council CoG - BioDisOrder 81964