Daily compared with alternate-day levamisole in pediatric nephrotic syndrome: an open-label randomized controlled study.

BACKGROUND: Levamisole is less expensive and has a better toxicity profile compared to other steroid sparing agents used in nephrotic syndrome. It has a plasma half-life of 2.0 to 5.6 hours, but is conventionally administered on alternate days. We aimed to assess whether daily levamisole is safe and more effective than standard alternate-day therapy in maintaining remission in children with frequently relapsing or steroid-dependent nephrotic syndrome (FR/SDNS). METHODS: An open-label randomized controlled trial was conducted in children with FR/SDNS. Group A received daily while Group B received alternate-day levamisole (2-3 mg/kg/dose) for 12 months. Prednisolone was tapered off by 3 months. Patients were monitored for relapses, further steroid requirement, and adverse effects. RESULTS: A total of 190 children with FR/SDNS (94 in Group A and 96 in Group B) were analyzed. Sustained remission for 12 months was observed in 36% of Group A and 27% of Group B patients (p = 0.18). Numbers completing 12 months in the study were 67% in Group A and 56% in Group B (p = 0.13). Time to first relapse, persistent FR/SDNS, and withdrawal due to poor compliance were statistically similar in both groups, while relapse rate and cumulative steroid dosage were significantly lower in Group A compared to Group B (p = 0.03 and p = 0.02, respectively). The incidence of adverse effects was comparable in both groups, with reversible leucopenia and hepatic transaminitis being the commonest. CONCLUSIONS: Daily levamisole therapy was not superior to alternate-day therapy in maintaining sustained remission over 12 months. Nevertheless, relapse rate and cumulative steroid dosage were significantly lower without increased adverse effects.

Antineutrophil cytoplasmic antibody in children with nephrotic syndrome treated with levamisole: a cross-sectional cohort study.

BACKGROUND: Levamisole is a commonly used steroid-sparing agent (SSA), but the reported incidence of antineutrophil cytoplasmic antibody (ANCA) positivity has been concerning. METHODS: Observational cross-sectional study wherein children aged 2 to 18 years with frequently relapsing/steroid dependent nephrotic syndrome (FRNS/SDNS) on levamisole for ≥ 12 months were tested for ANCA. RESULTS: A total of 210 children (33% female), median age of 7.3 (IQR: 5.6-9.6) years, and a median duration of levamisole exposure of 21 (IQR: 15-30) months were tested. ANCA was positive in 18% (n = 37): 89% (n = 33) perinuclear ANCA (pANCA), 3% (n = 1) cytoplasmic ANCA (cANCA), and 8% (n = 3) both. Of ANCA-positive children, none had reduced eGFR or abnormal urinalysis. The majority of these children were asymptomatic (81%, n = 30). Rash was more common among ANCA-positive children [6/37 (16%) vs. 3/173 (2%), p = 0.0001]. On multivariate analysis, higher age (OR = 1.02, [95th CI: 1.01 to 1.03], p = 0.007) and longer duration of levamisole exposure (OR = 1.05, [95th CI: 1.02 to 1.08], p = 0.0007) were associated with ANCA positivity. Levamisole was stopped in ANCA-positive children with the resolution of any clinical manifestations if present. Repeat ANCA testing was performed in 54% (20/37), and all were ANCA negative by 18 months. CONCLUSIONS: Children with FRNS/SDNS on longer duration of levamisole were associated with increasing prevalence of ANCA positivity, but most of these children were clinically asymptomatic. Prospective studies are required to determine the chronology of ANCA positivity and its clinical implication.

The clinical relevance of native vitamin D in pediatric kidney disease.

Hypovitaminosis D has been reported to be common in chronic kidney disease (CKD) as well as in proteinuric disorders. We reviewed available evidence to assess clinically relevant effects of low vitamin D status and native vitamin D (NVD) therapy, in pediatric renal diseases. Online medical databases were searched for articles related to vitamin D status, associations of hypovitaminosis D and effects of NVD therapy in kidney disease. Hypovitaminosis D was associated with worse skeletal, cardiovascular, inflammatory, and renal survival outcomes in CKD. Low serum 25 hydroxy-vitamin D (25[OH]D) levels correlated positively with glomerular filtration rate and negatively with serum parathyroid (PTH) levels. However, to date, evidence of benefit of NVD supplementation is restricted mainly to improvements in serum PTH, and biochemical 25[OH]D targets form the basis of clinical practice recommendations for NVD therapy. In nephrotic syndrome (NS) relapse, studies indicate loss of 25[OH]D along with vitamin D binding protein in urine, and serum total 25[OH]D levels are low. Preliminary evidence indicates that free 25[OH]D may be a better guide to the biologically active fraction. NVD therapy in NS does not show consistent results in improving skeletal outcomes and hypercalciuria has been reported when total 25[OH]D levels were considered as indication for therapy. NVD formulations should be regularised, and therapy monitored adequately to avoid adverse effects.

Ligand-Dependent Modulation of the Dynamics of Intracellular Loops Dictates Functional Selectivity of 5-HT2AR.

The serotonin 2A receptor (5-HT2AR) subtype of the G protein-coupled receptor (GPCR) family is involved in a plethora of neuromodulatory functions (e.g., neurogenesis, sleep, and cognitive processes). 5-HT2AR is the target of pharmacologically distinct classes of ligands, binding of which either activate or inactivate the receptor. Although high-resolution structures of 5-HT2AR as well as several other 5-HT GPCRs provided snapshots of both active and inactive conformational states, these structures, representing a truncated form of the receptor, cannot fully explain the mechanism of conformational transitions during their function. Importantly, biochemical studies have suggested the importance of intracellular loops in receptor functions. In our previous study, a model of the ligand-free form of 5-HT2AR with the third intracellular loop (ICL3) has been meticulously built. Here, we have investigated the functional regulation of 5-HT2AR with intact intracellular loops in ligand-free and five distinct ligand-bound configurations using unbiased atomistic molecular dynamics (MD) simulations. The selected ligands belong to either of the full, partial, or inverse agonist classes, which exert distinct pharmacological responses. We have observed significant structural, dynamic, and thermodynamic differences within ligand-bound complexes. Our results revealed, for the first time, that either activation or inactivation of the receptor upon specific ligand binding is primarily achieved through conformational transitions of its second and third intracellular loops (ICL2 and ICL3). A remarkable allosteric cross-talk between the ligand-binding site and the distal intracellular parts of the receptor, where binding of a specific ligand thermodynamically controls (either stabilizes or destabilizes) the intracellular region, consisting of crucial dynamic elements ICL2 and ICL3, and differential conformational transitions of these loops determine ligand-dependent functional selectivity.

Hidden electrostatic energy contributions define dynamic allosteric communications within p53 during molecular recognition.

Molecular recognition is fundamental to transcription regulation. As a transcription factor, the tumor suppressor p53 has to recognize either specific DNA sequences or repressor protein partners. However, the molecular mechanism underlying the p53 conformational switch from the DNA-bound to repressor-bound states is not fully characterized. The highly charged nature of these interacting molecules prompted us to explore the nonbonded energy contributions behind molecular recognition of either a DNA or the repressor protein iASPP by p53 DNA binding domain (p53DBD), using molecular dynamics simulation followed by rigorous analyses of energy terms. Our results illuminate the allosteric pathway by which iASPP binding to p53 diminishes binding affinity between p53 and DNA. Even though the p53DBD uses a common framework of residues for recognizing both DNA and iASPP, a comparison of the electrostatics in the two p53DBD complexes revealed significant differences in residue-wise contributions to the electrostatic energy. We found that an electrostatic allosteric communication path exists in the presence of both substrates. It consists of evolutionarily conserved residues, from residue K120 of the binding loop L1 to a distal residue R213 of p53DBD. K120 is near the DNA in the p53DBD-DNA complex, whereas iASPP binding moves it away from its DNA binding position in the p53DBD-iASPP complex. The "energy hubs" (the residues show a higher degree of connectivity with other residues in the electrostatic networks) determined from the electrostatic network analysis established that this conformational change in K120 completely rewires the electrostatic network from K120 to R213, thereby impeding DNA binding. Furthermore, we found shifting populations of hydrogen bonds and salt bridges reduce pairwise electrostatic energies within p53DBD in its DNA-bound state.

Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form.

The serotonin 2A receptor (5-HT2A R) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT2A R with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT2A R has marked distinction with ligand-bound conformations of 5-HT2 subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT2A R by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.

Retrospect and Prospect of Single Particle Cryo-Electron Microscopy: The Class of Integral Membrane Proteins as an Example.

A giant technological leap in the field of cryo-electron microscopy (cryo-EM) has assured the achievement of near-atomic resolution structures of biological macromolecules. As a recognition of this accomplishment, the Nobel Prize in Chemistry was awarded in 2017 to Jacques Dubochet, Joachim Frank, and Richard Henderson, the pioneers in the field of cryo-EM. Currently, the technique has become the method of choice for structural analysis of heterogeneous and intrinsically dynamic biological complexes. In the past few years, the most prolific branch of cryo-EM, single particle analysis, has revolutionized the structural biology field and structural investigation of membrane proteins, in particular. To achieve high-resolution structures of macromolecules in noncrystalline specimens, from sample and grid preparation to image acquisition, image data processing, and analysis of 3D maps, methodological advances in each of the steps play critical roles. In this Review, we discuss two areas in single particle cryo-EM, the remarkable developments in sample preparation strategies, particularly for membrane proteins, and breakthroughs in methodologies for molecular model building on the high-resolution 3D density maps that brought promises to resolve high-resolution (<4 Å) structures of biological macromolecules.

Resveratrol as a nontoxic excipient stabilizes insulin in a bioactive hexameric form.

Insulin aggregation is the leading cause of considerable reduction in the amount of active drug molecules in liquid formulations manufactured for diabetes management. Phenolic compounds, such as phenol and m-cresol, are routinely used to stabilize insulin in a hexameric form during its commercial preparation. However, long term usage of commercial insulin results in various adverse secondary responses, for which toxicity of the phenolic excipients is primarily responsible. In this study we aimed to find out a nontoxic insulin stabilizer. To that end, we have selected resveratrol, a natural polyphenol, as a prospective nontoxic insulin stabilizer because of its structural similarity with commercially used phenolic compounds. Atomic force microscopy visualization of resveratrol-treated human insulin revealed that resveratrol has a unique ability to arrest hINS in a soluble oligomeric form having discrete spherical morphology. Most importantly, resveratrol-treated insulin is nontoxic for HepG2 cells and it effectively maintains low blood glucose in a mouse model. Cryo-electron microscopy revealed 3D morphology of resveratrol-stabilized insulin that strikingly resembles crystal structures of insulin hexamer formulated with m-cresol. Significantly, we found that, in a condition inductive to amyloid fibrillation at physiological pH, resveratrol is capable of stabilizing insulin more efficiently than m-cresol. Thus, this study describes resveratrol as an effective nontoxic natural molecule that can be used for stabilizing insulin in a bioactive oligomeric form during its commercial formulation.

Free vitamin D levels in steroid-sensitive nephrotic syndrome and healthy controls.

INTRODUCTION: Body stores of vitamin D are measured as "total" serum 25-hydroxy vitamin D (25(OH)D). Its largest component is protein bound and lost in urine in nephrotic syndrome (NS). Our study investigates whether "free" 25(OH)D levels are a better guide to bone health and need for vitamin D supplementation in patients with steroid-sensitive NS (SSNS). METHODS: A cross-sectional study was performed in children with SSNS and healthy controls. Blood was tested for albumin, creatinine, calcium, phosphate, ALP, total and free (by direct ELISA) 25(OH)D, iPTH, and urine for protein-creatinine ratio. RESULTS: Seventy-nine NS patients (48 in relapse, 31 in remission) and 60 healthy controls were included. The levels of total 25(OH)D were significantly different (lowest in NS relapse and highest in controls) (p < 0.001). Corrected calcium and phosphate levels were normal, and there were no differences in free 25(OH)D, ALP, or iPTH levels between groups. Only total and not free 25(OH)D correlated significantly and negatively with urinary protein creatinine ratios (rs = - 0.42 vs. 0.04). Free 25(OH)D values of 3.75 and 2.85 pg/ml corresponded to total 25(OH)D levels of 20 and 12 ng/ml, respectively, in healthy controls. CONCLUSION: These results confirm that total 25(OH)D levels are low in NS and related to degree of proteinuria. However levels of free 25(OH)D, ALP, and iPTH did not change in relapse or remission in comparison with healthy controls. Our results suggest that in proteinuric renal diseases, free 25(OH)D rather than total 25(OH)D levels should be used to diagnose vitamin D deficiency and guide therapy.

Tumor Suppressor p53-Mediated Structural Reorganization of the Transcriptional Coactivator p300.

Transcriptional coactivator p300, a critical player in eukaryotic gene regulation, primarily functions as a histone acetyltransferase (HAT). It is also an important player in acetylation of a number of nonhistone proteins, p53 being the most prominent one. Recruitment of p300 to p53 is pivotal in the regulation of p53-dependent genes. Emerging evidence suggests that p300 adopts an active conformation upon binding to the tetrameric p53, resulting in its enhanced acetylation activity. As a modular protein, p300 consists of multiple well-defined domains, where the structured domains are interlinked with unstructured linker regions. A crystal structure of the central domain of p300 encompassing Bromo, RING, PHD, and HAT domains demonstrates a compact module, where the HAT active site stays occluded by the RING domain. However, although p300 has a significant role in mediating the transcriptional activity of p53, only a few structural details on the complex of these two full-length proteins are available. Here, we present a cryo-electron microscopy (cryo-EM) study on the p300-p53 complex. The three-dimensional cryo-EM density map of the p300-p53 complex, when compared to the cryo-EM map of free p300, revealed that substantial change in the relative arrangement of Bromo and HAT domains occurs upon complex formation, which is likely required for exposing HAT active site and subsequent acetyltransferase activity. Our observation correlates well with previous studies showing that the presence of Bromodomain is obligatory for effective acetyltransferase activity of HAT. Thus, our result sheds new light on the mechanism whereby p300, following binding with p53, gets activated.

Structural modules of the stress-induced protein HflX: an outlook on its evolution and biological role.

Multifunctional proteins often show modular structures. A functional domain and the structural modules within the domain show evolutionary conservation of their spatial arrangement since that gives the protein its functionality. However, the question remains as to how members of different domains of life (Archaea, Bacteria, Eukarya), polish and perfect these modules within conserved multidomain proteins, to tailor functional proteins according to their specific requirements. In the quest for plausible answers to this question, we studied the bacterial protein HflX. HflX is a universally conserved member of the Obg-GTPase superfamily but its functional role in Archaea and Eukarya is barely known. It is a multidomain protein and possesses, in addition to its conserved GTPase domain, an ATP-binding N-terminal domain. It is involved in heat stress response in Escherichia coli and our laboratory recently identified an ATP-dependent RNA helicase activity of E. coli HflX, which is likely instrumental in rescuing ribosomes during heat stress. Because perception and response to stress is expected to be different in different life forms, the question is whether this activity is preserved in higher organisms or not. Thus, we explored the evolution pattern of different structural modules of HflX, with particular emphasis on the ATP-binding domain, to understand plausible biological role of HflX in other forms of life. Our analyses indicate that, while the evolutionary pattern of the GTPase domain follows a conserved phylogeny, conservation of the ATP-binding domain shows a complicated pattern. The limited analysis described here hints towards possible evolutionary adaptations and modifications of the domain, something which needs to be investigated in more depth in homologs from other life forms. Deciphering how nature 'tweaks' such modules, both structurally and functionally, may help in understanding the evolution of such proteins, and, on a large-scale, of stress-related proteins in general as well.

The effect of vitamin D and calcium supplementation in pediatric steroid-sensitive nephrotic syndrome.

BACKGROUND: Low serum levels of total 25-hydroxycholecalciferol (25(OH)D) occur in nephrotic syndrome (NS). We aimed to assess the effects of vitamin D3 and calcium supplementation on 25(OH)D levels, bone mineralization, and NS relapse rate in children with steroid-sensitive NS. METHODS: A randomized controlled trial (RCT) was performed in children with steroid-sensitive NS. The treatment group received vitamin D3 (60,000 IU orally, weekly for 4 weeks) and calcium supplements (500 to 1,000 mg/day for 3 months) after achieving NS remission. Blood samples for bone biochemistry were taken during relapse (T0), after 6 weeks (T1) and 6 months (T2) of randomization, whereas a lumbar DXA scan was performed at T0 and T2. Renal ultrasound was performed after study completion in the treatment group and in all patients with hypercalciuria. RESULTS: Of the 48 initial recruits, 43 patients completed the study. Post-intervention, 25(OH)D levels showed significant improvements in the treatment group compared with controls at T1 (p < 0.001) and T2 (p < 0.001). However, this was not associated with differences in bone mineral content (BMC) (p = 0.44) or bone mineral density (BMD) (p = 0.64) between the groups. Additionally, there was no reduction in relapse number in treated patients (p = 0.54). Documented hypercalciuria occurred in 52% of patients in the treatment group, but was not associated with nephrocalcinosis. CONCLUSIONS: Although supplementation with calcium and vitamin D improved 25(OH)D levels significantly, there was no effect on BMC, BMD or relapse rate over a 6-month follow-up. Occurrence of hypercalciuria mandates caution and appropriate monitoring if using such therapy. Appropriate dosage of vitamin D3 remains uncertain and studies examining biologically active vitamin D may provide answers.

Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein.

It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded. In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg(+2) ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg(+2) ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing. For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

Cryo-EM structures reveal the molecular mechanism of HflX-mediated erythromycin resistance in mycobacteria.

Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M. smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX's ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria.

Structural insights into the mechanism of translational inhibition by the fungicide sordarin.

The translational machinery has been found to be the target for a number of antibiotics. One such antibiotic sordarin selectively inhibits fungal translation by impairing the function of elongation factor 2 (eEF2) while being ineffective to higher eukaryotes. Surprisingly, sordarin is not even equally effective in impairing translation for all fungal species. The binding cavity of sordarin on eEF2 has been localized by X-ray crystallographic study and its unique specificity towards sordarin has been attributed to the species specific substitutions within a stretch of amino acids (sordarin specificity region, SSR) at the entrance of the cavity. In this study, we have analyzed the sordarin-binding cavity of eEF2 from different species both in isolated and ribosome-bound forms in order to decipher the mechanism of sordarin binding selectivity. Our results reveal that the molecular architecture as well as the microenvironment of the sordarin-binding cavity changes significantly from one species to another depending on the species specific substitutions within the cavity. Moreover, eEF2 binding to ribosome aggravates the effects of these substitutions. Thus, this study, while shedding light on the molecular mechanism underpinning the selective inhibitory effects of sordarin, will also be a helpful guide for future studies aiming at developing novel antifungal drugs with broader spectrum of activity.

Vitamin D in nephrotic syndrome remission: a case-control study.

BACKGROUND: Vitamin D deficiency may contribute to osteoporosis in nephrotic syndrome (NS). METHODS: A cross-sectional case-control study was performed to investigate 25 hydroxycholecalciferol [25(OH)D] status in 40 patients with NS in remission and 40 healthy controls. Serum levels of 25(OH)D, calcium, phosphate, alkaline phosphatase (ALP), and intact parathyroid hormone (PTH) were assayed. NS patients were segregated by age at onset, current age, type and duration of NS, months since relapse and current drug therapy. RESULTS: Levels of 25(OH)D showed a positive correlation with months elapsed since last NS relapse (r s = +0.4, p = 0.012) and were lower in NS patients within 3 months of relapse but similar to that of controls in patients in remission for >3 months [median 14.23 (interquartile range 12.19-17.63) vs. 19.75 (14.04-28.38) ng/ml, respectively; p = 0.039]. There was no correlation of 25(OH)D levels with other disease characteristics or drug therapy. ALP levels were also lowest after relapse (r s = +0.34, p = 0.036). Overall, 25(OH)D levels of <20 ng/ml occurred in 62.5 % of NS patients + controls, and correlated negatively with age (r s = -0.24, p = 0.037) but showed no significant correlation with calcium, phosphate, or PTH levels. CONCLUSIONS: In our patients with NS, vitamin D stores remained low for 3 months after NS relapse but showed an increase with longer remission time to control levels. Vitamin D stores were not influenced by disease characteristics or therapy. Longitudinal studies are required to confirm these findings and evaluate the effect of vitamin D on bones, particularly in frequent relapsers.

Outcome of severe steroid-dependent nephrotic syndrome treated with mycophenolate mofetil.

BACKGROUND: Mycophenolate mofetil (MMF) is used as a steroid-sparing agent in pediatric nephrotic syndrome (NS). However, data about its long-term efficacy and safety is limited. METHODS: We report the long-term outcome of MMF therapy in 46 NS patients who remained steroid dependent (SD) despite previous treatment with levamisole and cyclophosphamide. RESULTS: After 1 year of MMF initiation, 32 (70 %) patients had reduced steroid requirement: 12 with decreased threshold dose and 20 were able to stop steroids. At follow-up of mean 3.56 (standard deviation + 1.76) years, 25 (54 %) children required no further alternative immunosuppression (IS), having infrequent or no relapses, of which 14 stopped MMF after a mean 2.4 (standard deviation + 0.9) years; 11 are continuing on MMF for a median of 2.25 years (range 1.33-7.75 years). One patient had a psoriasis flare, and MMF was stopped. No other patient required permanent drug withdrawal due to side effects. The outcome of patients who did not require further alternate IS was significantly better than those who did, with 56 % vs. 10.5 %, respectively, being off regular medications at last follow-up. CONCLUSIONS: We conclude that MMF therapy is safe in the long term and allows >50 % of severe SDNS patients to avoid further toxic IS.

Cryo-EM captures a unique conformational rearrangement in 23S rRNA helices of the Mycobacterium 50S subunit.

Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report structural evidence of a hitherto unknown conformational switch of mycobacterium 23S rRNA helices (H54a and H67-H71). Cryo-electron microscopy (cryo-EM) structures (~3-4 Å) of the M. smegmatis (Msm) log-phase 50S ribosomal subunit revealed conformational variability in H67-H71 region of the 23S rRNA, and manifested that, while H68 possesses the usual stretched conformation in one class of the maps, another one exhibits a bulge-out, fused density of H68-H69 at the inter-subunit surface, indicating an intrinsic dynamics of these rRNA helices. Remarkably, altered conformation of H68 forming a more prominent bulge-out structure at the inter-subunit surface of the 50S subunit due to the conformational rearrangements of 23S rRNA H67-H71 region was clearly visualized in a 3 Å cryo-EM map of the 50S subunit obtained from the stationary phase ribosome dataset. The Msm50S subunit having such bulge-out conformation at the intersubunit surface would be incompatible for associating with the 30S subunit due to its inability to form major inter-subunit bridges. Evidently, availability of active 70S ribosome pool can be modulated by stabilizing either one of the H68 conformation.

Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis.

In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-A cryo-electron microscopy map of the aminoacyl-tRNA x EF-Tu x GDP x kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism.

The role of membranes in function and dysfunction of intrinsically disordered amyloidogenic proteins.

Membrane-protein interactions play a major role in human physiology as well as in diseases pathology. Interaction of a protein with the membrane was previously thought to be dependent on well-defined three-dimensional structure of the protein. In recent decades, however, it has become evident that a large fraction of the proteome, particularly in eukaryotes, stays disordered in solution and these proteins are termed as intrinsically disordered proteins (IDPs). Also, a vast majority of human proteomes have been reported to contain substantially long disordered regions, called intrinsically disordered regions (IDRs), in addition to the structurally ordered regions. IDPs exist in an ensemble of conformations and the conformational flexibility enables IDPs to achieve functional diversity. IDPs (and IDRs) are found to be important players in cell signaling, where biological membranes act as anchors for signaling cascades. Therefore, IDPs modulate the membrane architectures, at the same time membrane composition also affects the binding of IDPs. Because of intrinsic disorders, misfolding of IDPs often leads to formation of oligomers, protofibrils and mature fibrils through progressive self-association. Accumulation of amyloid-like aggregates of some of the IDPs is a known causative agent for numerous diseases. In this chapter we highlight recent advances in understanding membrane interactions of some of the intrinsically disordered proteins involved in the pathogenesis of human diseases.