Stabilization of pre-existing neurotensin receptor conformational states by ß-arrestin-1 and the biased allosteric modulator ML314.

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13CεH3-methionine NMR spectroscopy to show that binding of phosphatidylinositol-4,5-bisphosphate (PIP2) to the receptor's intracellular surface allosterically tunes the timescale of motions at the orthosteric pocket and conserved activation motifs - without dramatically altering the structural ensemble. ß-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on exchange rates. A ß-arrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Ligands selectively tune the local and global motions of neurotensin receptor 1 (NTS1).

Nuclear magnetic resonance (NMR) studies have revealed that fast methyl sidechain dynamics can report on entropically-driven allostery. Yet, NMR applications have been largely limited to the super-microsecond motional regimes of G protein-coupled receptors (GPCRs). We use 13Cε-methionine chemical shift-based global order parameters to test if ligands affect the fast dynamics of a thermostabilized GPCR, neurotensin receptor 1 (NTS1). We establish that the NTS1 solution ensemble includes substates with lifetimes on several, discrete timescales. The longest-lived states reflect those captured in agonist- and inverse agonist-bound crystal structures, separated by large energy barriers. We observe that the rapid fluctuations of individual methionine residues, superimposed on these long-lived states, respond collectively with the degree of fast, global dynamics correlating with ligand pharmacology. This approach lends confidence to interpreting spectra in terms of local structure and methyl dihedral angle geometry. The results suggest a role for sub-microsecond dynamics and conformational entropy in GPCR ligand discrimination.

A Methionine Chemical Shift Based Order Parameter Characterizing Global Protein Dynamics.

Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The 13 C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.

Cis-trans proline isomers in the catalytic domain of calcineurin.

Calcineurin is an essential calcium-activated serine/threonine phosphatase. The six NMR-observable methionine methyl groups in the catalytic domain of human calcineurin Aα (CNA) were assigned and used as reporters of the presence of potential cis-trans isomers in solution. Proline 84 is found in the cis conformation in most calcineurin X-ray structures, and proline 309, which is part of a highly conserved motif in phosphoprotein phosphatases, was modeled with a cis peptide bond in one of the two molecules present in the asymmetric unit of CNA. We mutated each of the two prolines to alanine to force the trans conformation. Solution NMR shows that the P84A CNA mutant exists in two forms, compatible with cis-trans isomers, while the P309A mutant is predominantly in the trans conformation. DATABASE: PDB depositions mentioned PDB 5C1V and 2JOG.

Appropriateness of lumbar spine magnetic resonance imaging in Spain.

OBJECTIVES: To determine the minimum percentage of lumbar spine magnetic resonance imaging (LSMRI) which are inappropriately prescribed in routine practice. METHODS: LSMRI performed prospectively on 602 patients in 12 Radiology Services across 6 regions in Spain, were classified as "appropriate", "uncertain" or "inappropriate" based on the indication criteria established by the National Institute for Clinical Excellence, the American College of Physicians and Radiology, and current evidence-based clinical guidelines. Studies on patients reporting at least one "red flag" were classified as "appropriate". A logistic regression model was developed to identify factors associated with a higher likelihood of inappropriate LSMRI, including gender, reporting of referred pain, health care setting (private/public), and specialty of prescribing physician. Before performing the LSMRI, the radiologists also assessed the appropriateness of the prescription. RESULTS: Eighty-eight percent of LSMRI were appropriate, 1.3% uncertain and 10.6% inappropriate. The agreement of radiologists' assessment with this classification was substantial (k=0.62). The odds that LSMRI prescriptions were inappropriate were higher for patients without referred pain [OR (CI 95%): 13.75 (6.72; 28.16)], seen in private practice [2.25 (1.20; 4.22)], by orthopedic surgeons, neurosurgeons or primary care physicians [2.50 (1.15; 5.56)]. CONCLUSION: Efficiency of LSMRI could be improved in routine practice, without worsening clinical outcomes.

Correlation of the EPR properties of perchlorotriphenylmethyl radicals and their efficiency as DNP polarizers.

Water soluble perchlorinated trityl (PTM) radicals were found to be effective 95 GHz DNP (dynamic nuclear polarization) polarizers in ex situ (dissolution) (13)C DNP (Gabellieri et al., Angew Chem., Int. Ed. 2010, 49, 3360). The degree of the nuclear polarization obtained was reported to be dependent on the position of the chlorine substituents on the trityl skeleton. In addition, on the basis of the DNP frequency sweeps it was suggested that the (13)C NMR signal enhancement is mediated by the Cl nuclei. To understand the DNP mechanism of the PTM radicals we have explored the 95 GHz EPR characteristics of these radicals that are relevant to their performance as DNP polarizers. The EPR spectra of the radicals revealed axially symmetric g-tensors. A comparison of the spectra with the (13)C DNP frequency sweeps showed that although the solid effect mechanism is operational the DNP frequency sweeps reveal some extra width suggesting that contributions from EPR forbidden transitions involving (35,37)Cl nuclear flips are likely. This was substantiated experimentally by ELDOR (electron-electron double resonance) detected NMR measurements, which map the EPR forbidden transitions, and ELDOR experiments that follow the depolarization of the electron spin upon irradiation of the forbidden EPR transitions. DFT (density functional theory) calculations helped to assign the observed transitions and provided the relevant spin Hamiltonian parameters. These results show that the (35,37)Cl hyperfine and nuclear quadrupolar interactions cause a considerable nuclear state mixing at 95 GHz thus facilitating the polarization of the Cl nuclei upon microwave irradiation. Overlap of Cl nuclear frequencies and the (13)C Larmor frequency further facilitates the polarization of the (13)C nuclei by spin diffusion. Calculation of the (13)C DNP frequency sweep based on the Cl nuclear polarization showed that it does lead to an increase in the width of the spectra, improving the agreement with the experimental sweeps, thus supporting the existence of a new heteronuclear assisted DNP mechanism.

Polychlorinated trityl radicals for dynamic nuclear polarization: the role of chlorine nuclei.

Polychlorinated trityl radicals bearing carboxylate substituents are water soluble persistent radicals that can be used for dynamic nuclear polarization. In contrast to other trityl radicals, the polarization mechanism differs from the classical solid effect. DFT calculations performed to rationalize this behaviour support the hypothesis that polarization is transferred from the unpaired electron to chlorine nuclei and from these to carbon by spin diffusion. The marked differences observed between neutral and anionic forms of the radical will be discussed.

Hematoma espinal subdural espontáneo / Spontaneous spinal subdural hematoma

El hematoma espinal subdural es una patología extremadamente rara. Aparece normalmente asociado a punciones lumbares y/o a discrasias sanguíneas. Presentamos el caso de un varón de 78 años, con antecedente de toma de anticoagulantes orales que presentó de forma brusca un déficit neurológico motor de ambos miembros inferiores. Describimos los hallazgos en RM, que fueron confirmados posteriormente de forma quirúrgica. Destacamos la importancia que esta técnica tiene tanto en la identificación precoz de la colección hemática, para diferenciarla de otras patología espinales, ya que puede ser necesario un tratamiento quirúrgico inmediato, como en el seguimiento de estos pacientes, ya que es indispensable para la valoración de atrofia medular o signos de aracnoiditis

Spinal subdural hematoma is extremely rare. It is normally associated to lumbar puncture and/or blood disorders. We present the case of 78-year-old man with a history of oral anticoagulant treatment that presented with a sudden neurologic motor deficit in the lower limbs. We describe the MRI findings, which were confirmed at surgery. We emphasize the importance of MRI both in the early identification of the hematic collection, as immediate surgical treatment may be necessary, and in the follow-up of these patients, as it is essential in the evaluation of atrophy of the spinal cord and signs of arachnoiditis