Universal pathway for posttransfer editing reactions: Insights from the crystal structure of TtPheRS with puromycin

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA^Phe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA^Phe where deacylation occurs from either the 2′- or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.A key role in genetic code translation play aminoacyl-tRNA synthetases (aaRSs), providing linkage of amino acids to tRNAs. Before activation, at the amino acid recognition step, some aaRSs face a challenge of discrimination among amino acids with closely similar chemical structure. The rate of erroneous aminoacylation products generated in vivo is no more than one error per 10⁴−10⁵ correct reactions (1). To ensure such an extent of accuracy, aaRSs developed a multisieve mechanism of proofreading (2, 3). The existence of a proofreading activity has been demonstrated for both class I and class II aaRSs. Among aaRSs on record, about half of them are capable of selecting between amino acids resembling each other (4).The class aminoacyl-tRNA synthetases (aaRSs), namely IleRS, ValRS, and LeuRS, are characterized by a conserved connective polypeptide 1 (CP1) editing domain forming insertion into the catalytic core, except in cases of bacterial and mitochondrial LeuRSs, where the CP1 occurs at a different point of insertion (5). MetRS also falls into class I, but its CP1 lacks editing activity (6). The editing domains of class II aaRSs (ThrRS, ProRS, AlaRS, PheRS) are more diverse in amino acid sequence and in the distinguishing features of their folds. Kinetic experiments carried out for SerRS revealed the presence of a tRNA-independent pretransfer editing pathway (7).Detailed analyses of posttransfer editing were performed for class I LeuRS and class II ThrRS (8, 9). The structures of the LeuRS posttransfer complex imply the existence of water molecules that are specifically coordinated, to play the role of attacking nucleophiles. The alanine-scanning mutagenesis of the editing site has failed to identify key residues directly involved in catalysis (8). Thus, it was proposed that the CP1 domain simply binds the substrates in a configuration that favors attack by a water molecule, which itself is appropriately positioned by the set of key residues. The crystal structure of the editing domain from ThrRS complexed with Ser-A76 reveals two water molecules located on either side of the hydrolyzed bond (9). This study underlines the crucial role played by tRNA in substrate-assisted catalysis, in positioning the catalytic water molecules along with the protein side chains (9, 10).The 3D structures of Thermus thermophilus phenylalanyl-tRNA synthetase (TtPheRS) and its complexes with functional substrates (11–14) revealed that the catalytic α subunit exerts control over aminoacylation reaction whereas the major role of the β subunit lies in the recognition and binding of cognate tRNA^Phe and hydrolysis of misacylated tRNA (Fig. 1A). The early fast kinetic study demonstrated that tyrosine is indeed transferred to tRNA^Phe, and the misacylated tRNA is rapidly hydrolyzed (15). Later, it was established that editing activity of the bacterial and archaeal/eukaryotic PheRSs is associated with the active site located at the interface region between B3 and B4 domains in the β subunit (16–18).Open in a separate windowFig. 1.(A) Structure of the TtPheRS complex with puromycin and phenylalanine. The protein is shown in cartoon representation; the ligands puromycin (red) and phenylalanine (dark blue) are shown in space-filling representation. The domain architecture of one αβ-heterodimer is shown with the N-terminal coiled coil of the α-subunit colored cyan, catalytic domains A1 and A2 colored red, and structural domains of the β-subunit domains from B1 to B8 colored differently. The symmetry-related heterodimer is denoted with asterisks. (B) The editing site cavity of TtPheRS with bound puromycin. The electron density map (colored in red), calculated as described in SI Materials and Methods with coefficients (F_obs − F_calc) contoured at 2.5σ. Crystal structure of the TtPheRS complex in space-filling representation (colored gray) rendered to show protein surface interacting with puromycin.Here we present the crystal structure of TtPheRS, in complex with phenylalanine at the “synthetic” (aminoacylation) site and puromycin (mimicking the A76 of tRNA misacylated with Tyr) at the editing site. The natural substrate’s ester moiety represents an isoelectronic analog of the puromycin amide group, wherein the NH group is replaced with an ester oxygen atom. The appearance of puromycin at the editing site is accompanied by changes in the positions of some bound water molecules or even by their loss, compared with TtPheRS complex with Tyr (17). Loss of the water molecules, supposedly underlying the nucleophilic attack on the carbonyl carbon of the ester bond, gives grounds for revisiting the hydrolytic mechanism at work in bacterial PheRSs (17, 19). The suggested pathway of hydrolytic reaction is of sufficient generality to warrant comparison with those of other class I and class II aaRSs.     

Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

The tumor suppressor p53 is mutationally inactivated in ≈50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in ≈75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of ≈150 μM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein–PhiKan083 complex at 1.5-Å resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent “druggable” target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.     

Validation of surface‐to‐volume ratio measurements derived from oscillating gradient spin echo on a clinical scanner using anisotropic fiber phantoms

A diffusion measurement in the short‐time surface‐to‐volume ratio (S/V) limit (Mitra et al., Phys Rev Lett. 1992;68:3555) can disentangle the free diffusion coefficient from geometric restrictions to diffusion. Biophysical parameters, such as the S/V of tissue membranes, can be used to estimate microscopic length scales non‐invasively. However, due to gradient strength limitations on clinical MRI scanners, pulsed gradient spin echo (PGSE) measurements are impractical for probing the S/V limit. To achieve this limit on clinical systems, an oscillating gradient spin echo (OGSE) sequence was developed. Two phantoms containing 10 fiber bundles, each consisting of impermeable aligned fibers with different packing densities, were constructed to achieve a range of S/V values. The frequency‐dependent diffusion coefficient, D(ω), was measured in each fiber bundle using OGSE with different gradient waveforms (cosine, stretched cosine, and trapezoidal), while D(t) was measured from PGSE and stimulated‐echo measurements. The S/V values derived from the universal high‐frequency behavior of D(ω) were compared against those derived from quantitative proton density measurements using single spin echo (SE) with varying echo times, and from magnetic resonance fingerprinting (MRF). S/V estimates derived from different OGSE waveforms were similar and demonstrated excellent correlation with both SE‐ and MRF‐derived S/V measures (ρ ≥ 0.99). Furthermore, there was a smoother transition between OGSE frequency f and PGSE diffusion time when using , rather than the commonly used t_eff = 1/(4f), validating the specific frequency/diffusion time conversion for this regime. Our well‐characterized fiber phantom can be used for the calibration of OGSE and diffusion modeling techniques, as the S/V ratio can be measured independently using other MR modalities. Moreover, our calibration experiment offers an exciting perspective of mapping tissue S/V on clinical systems.     

Non-symmetrical double-logistic analysis of 24-h blood pressure recordings in normotensive and hypertensive rats

OBJECTIVE: To determine the suitability of a new logistic curve fitting procedure to measure the diurnal rates of transition from the active to the asleep periods separately. METHOD: We applied this method to 24-h telemetry recordings of systolic, mean, diastolic arterial pressure (SAP, MAP, DAP, respectively), heart rate (HR) and locomotor activity of normotensive Sprague-Dawley rats (SDR) and spontaneously hypertensive rats (SHR). RESULTS: There was a similar pattern of higher awake and lower sleep values (16 +/- 1 mmHg SAP, 77 +/- 2 bpm HR and 40 +/- 2 units activity) in SHR. In SDR, awake-asleep differences were less for SAP (9 +/- 1 mmHg) but similar for HR (83 +/- 2 bpm). In SHR, while the blood pressure patterns were symmetrical, the rate of rise in activity and HR during arousal was more rapid than the rate of decline during the dark to light transition. By contrast in SDR, the arousal rate of increase in blood pressure and HR was much less than the rate of decline. Thus SHR have an exaggerated arousal surge in DAP compared with SDR. Double logistic provides a better fit than Cosinor or square wave and better estimates of day-night differences than partial Fourier. CONCLUSIONS: Analysis of 24-h recordings by a new logistic curve method reveals distinct asymmetric circadian patterns of cardiovascular and activity changes in rats. The greater surge in arousal blood pressure in SHR is not associated with differences in HR or activity changes and may be inherent to the underlying mechanisms contributing to the hypertension in SHR.     

Clinical Scale Zinc Finger Nuclease-mediated Gene Editing of PD-1 in Tumor Infiltrating Lymphocytes for the Treatment of Metastatic Melanoma

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9–84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500–2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.     

Abnormal intrastore calcium signaling in chronic heart failure

Diminished Ca release from the sarcoplasmic reticulum (SR) is an important contributor to the impaired contractility of the failing heart. Despite extensive effort, the underlying causes of abnormal SR Ca release in heart failure (HF) remain unknown. We used a combination of simultaneous imaging of cytosolic and SR intraluminal [Ca] in isolated cardiomyocytes and recordings from single-ryanodine receptor (RyR) channels reconstituted into lipid bilayers to investigate alterations in intracellular Ca handling in an experimental model of chronic HF. We found that diastolic free [Ca] inside the SR was dramatically reduced because of a Ca leak across the SR membrane, mediated by spontaneous local release events (Ca sparks), in HF myocytes. Additionally, the magnitudes of intrastore Ca depletion signals during global and focal Ca release events were blunted, and [Ca]SR recovery was slowed after global but not focal Ca release in HF myocytes. At the single-RyR level, the sensitivity of RyRs to activation by luminal Ca was greatly enhanced, providing a molecular mechanism for the maintained potentiation of Ca sparks (and increased Ca leak) at reduced intra-SR [Ca] in HF. This work shows that the diminished SR Ca release characteristic of failing myocardium could be explained by increased sensitivity of RyRs to luminal Ca, leading to enhanced spark-mediated SR Ca leak and reduced intra-SR [Ca].     

Reconstruction by bone transport after resection of benign tumors of tibia: A retrospective study of 38 patients

Background:

The commonly used reconstructive options after post resection defects in bone tumors like megaprosthesis, autograft, allograft, bone graft substitutes and recycled bone have their own demerits on a long term. Bone transport that regenerates patient''s own bone is a less explored option of reconstruction after resection of benign bone tumors and reports on this are limited. This technique is very much relevant in tibia where Ilizarov fixator is surgeon and patient friendly. We report our experience.

Materials and Methods:

This is a retrospective series of resection and bone transport in 38 patients with benign tumor of tibia. There were 14 males and 24 females with mean age of 23.40 years (range 9–40 years). Lesion was located in proximal third tibia in 27, middle third in two and distal third in nine patients. The diagnosis was giant cell tumor in 32, chondroblastoma in three, chondromyxoid fibroma, enchondroma and desmoplasic fibroma in one patient each. The resection was intercalary in 28 and transarticular in 10 patients. Osteosynthesis was monofocal in three, bifocal in 31 and polyfocal in four cases.

Results:

Mean followup was 7.22 years (range 1.5–15 years). Mean resection length was 10.21 cm (range 3–22 cm). The mean duration of external fixator was 308.03 days (range 89–677 days) and mean external fixator index was 36.14 days/cm (range 16.84–97.43 days/cm). Twelve patients had difficulties in the form of 11 problems and five obstacles that were successfully managed. None of the patients had local recurrence of tumor or any long term complication. Mean Musculo-skeletal Tumour Society score at final followup was 27.18 (90.60%).

Conclusions:

Bone transport is an excellent option after resection of benign tumors of tibia with good local control and functional outcome, despite minor difficulties that need timely management.