Qualification of a LC-HRMS platform method for biosimilar development using NISTmab as a model.

Biosimilars are a cost-effective alternative to biopharmaceuticals, necessitating rigorous analytical methods for consistency and compliance. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is a versatile tool for assessing key attributes, encompassing molecular mass, primary structure, and post-translational modifications (PTMs). Adhering to ICH Q2R1, we validated an LC-HRMS based peptide mapping method using NISTmab as a reference. The method validation parameters, covering system suitability, specificity, accuracy, precision, robustness, and carryover, were comprehensively assessed. The method effectively differentiated the NISTmab from similar counterparts as well as from artificially introduced spiked conditions. Notably, the accuracy of mass error for NISTmab specific complementarity determining region peptides was within a maximum of 2.42 parts per million (ppm) from theoretical and the highest percent relative standard deviation (%RSD) observed for precision was 0.000219 %. It demonstrates precision in sequence coverage and PTM detection, with a visual inspection of total ion chromatogram approach for variability assessment. The method maintains robustness when subjected to diverse storage conditions, encompassing variations in column temperature and mobile phase composition. Negligible carryover was noted during the carryover analysis. In summary, this method serves as a versatile platform for multiple biosimilar development by effectively characterizing and identifying monoclonal antibodies, ultimately ensuring product quality.

Unraveling the stereochemical and dynamic aspects of the catalytic site of bacterial peptidyl-tRNA hydrolase.

Bacterial peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) hydrolyzes the peptidyl-tRNAs accumulated in the cytoplasm and thereby prevents cell death by alleviating tRNA starvation. X-ray and NMR studies of Vibrio cholerae Pth (VcPth) and mutants of its key residues involved in catalysis show that the activity and selectivity of the protein depends on the stereochemistry and dynamics of residues H24, D97, N118, and N14. D97-H24 interaction is critical for activity because it increases the nucleophilicity of H24. The N118 and N14 have orthogonally competing interactions with H24, both of which reduce the nucleophilicity of H24 and are likely to be offset by positioning of a peptidyl-tRNA substrate. The region proximal to H24 and the lid region exhibit slow motions that may assist in accommodating the substrate. Helix α3 exhibits a slow wobble with intermediate time scale motions of its N-cap residue N118, which may work as a flypaper to position the scissile ester bond of the substrate. Overall, the dynamics of interactions between the side chains of N14, H24, D97, and N118, control the catalysis of substrate by this enzyme.

Structural and functional characterization of the transcriptional regulator Rv3488 of Mycobacterium tuberculosis H37Rv.

Rv3488 of Mycobacterium tuberculosis H37Rv has been assigned to the phenolic acid decarboxylase repressor (PadR) family of transcriptional regulators that play key roles in multidrug resistance and virulence of prokaryotes. The binding of cadmium, zinc, and several other metals to Rv3488 was discovered and characterized by isothermal titration calorimetery to be an exothermic process. Crystal structures of apo-Rv3488 and Rv3488 in complex with cadmium or zinc ions were determined by X-ray crystallography. The structure of Rv3488 revealed a dimeric protein with N-terminal winged-helix-turn-helix DNA-binding domains composed of helices α1, α2, α3, and strands ß1 and ß2, with the dimerization interface being formed of helices α4 and α1. The overall fold of Rv3488 was similar to PadR-s2 and metal sensor transcriptional regulators. In the crystal structure of Rv3488-Cd complex, two octahedrally coordinated Cd2+ ions were present, one for each subunit. The same sites were occupied by zinc ions in the structure of Rv3488-Zn, with two additional zinc ions complexed in one monomer. EMSA studies showed specific binding of Rv3488 with its own 30-bp promoter DNA. The functional role of Rv3488 was characterized by expressing the rv3488 gene under the control of hsp60 promoter in Mycobacterium smegmatis Expression of Rv3488 increased the intracellular survival of recombinant M. smegmatis in murine macrophage cell line J774A.1 and also augmented its tolerance to Cd2+ ions. Overall, the studies show that Rv3488 may have transcription regulation and metal-detoxifying functions and its expression in M. smegmatis increases intracellular survival, perhaps by counteracting toxic metal stress.

Structural and Biophysical Characterization of Rab5a from Leishmania Donovani.

Leishmania donovani possess two isoforms of Rab5 (Rab5a and Rab5b), which are involved in fluid phase and receptor-mediated endocytosis, respectively. We have characterized the solution structure and dynamics of a stabilized truncated LdRab5a mutant. For the purpose of NMR structure determination, protein stability was enhanced by systematically introducing various deletions and mutations. Deletion of hypervariable C-terminal and the 20 residues LdRab5a specific insert slightly enhanced the stability, which was further improved by C107S mutation. The final construct, truncated LdRab5a with C107S mutation, was found to be stable for longer durations at higher concentration, with an increase in melting temperature by 10°C. Solution structure of truncated LdRab5a shows the characteristic GTPase fold having nucleotide and effector binding sites. Orientation of switch I and switch II regions match well with that of guanosine 5'-(ß, γ-imido)triphosphate (GppNHp)-bound human Rab5a, indicating that the truncated LdRab5a attains the canonical GTP bound state. However, the backbone dynamics of the P-loop, switch I, and switch II regions were slower than that observed for guanosine 5'-(ß, γ-imido)triphosphate (GMPPNP)-bound H-Ras. This dynamic profile may further complement the residue-specific complementarity in determining the specificity of interaction with the effectors. In parallel, biophysical investigations revealed the urea induced unfolding of truncated LdRab5a to be a four-state process that involved two intermediates, I1 and I2. The maximal 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding was observed for I2 state, which was inferred to have molten globule like characteristics. Overall, the strategy presented would have significant impact for studying other Rab and small GTPase proteins by NMR spectroscopy.

Solution structure and dynamics of glia maturation factor from Caenorhabditis elegans.

BACKGROUND: The GMF class of the ADF-H domain family proteins regulate actin dynamics by binding to the Arp2/3 complex and F-actin through their Site-1 and Site-2, respectively. CeGMF of C. elegans is analogous to GMFγ of human and mouse and is 138 amino acids in length. METHODS: We have characterized the solution structure and dynamics of CeGMF by solution NMR spectroscopy and its thermal stability by DSC. RESULTS: The solution structure of CeGMF shows canonical ADF-H fold with two additional ß-strands in the ß4-ß5 loop region. The Site-1 of CeGMF is well formed and residues of all three regions of Site-1 show dynamic flexibility. However, the ß4-ß5 loop of Site-2 is less inclined towards the C-terminal, as the latter is truncated by four residues in comparison to GMF isoforms of human and mouse. Regions of Site-2 show motions on ns-ps timescale, but dynamic flexibility of ß4-ß5 loop is low in comparison to corresponding F-loop region of ADF/cofilin UNC-60B. A general difference in packing of α3 and α1 between GMF and ADF/cofilins was noticed. Additionally, thermal stability of CeGMF was significantly higher than its ADF/cofilin homologs. CONCLUSION: We have presented the first solution structure of GMF from C. elegans, which highlights the structural differences between the Site-2 of CeGMF and mammalian GMF isoforms. Further, we have seen the differences in structure, dynamics, and thermal stability of GMF and ADF/cofilin. GENERAL SIGNIFICANCE: This study provides a useful insight to structural and dynamics factors that define the specificity of GMF towards Arp2/3 complex.

Role of methionine 71 in substrate recognition and structural integrity of bacterial peptidyl-tRNA hydrolase.

BACKGROUND: Bacterial peptidyl-tRNA hydrolase (Pth) is an essential enzyme that alleviates tRNA starvation by recycling prematurely dissociated peptidyl-tRNAs. The specificity of Pth for N-blocked-aminoacyl-tRNA has been proposed to be contingent upon conserved residue N14 forming a hydrogen bond with the carbonyl of the first peptide bond in the substrate. M71 is involved in forming a conserved hydrogen bond with N14. Other interactions facilitating this recognition are not known. METHODS: The structure, dynamics, and stability of the M71A mutant of Pth from Vibrio cholerae (VcPth) were characterized by X-ray crystallography, NMR spectroscopy, MD simulations and DSC. RESULTS: Crystal structure of M71A mutant was determined. In the structure, the dimer interface is formed by the insertion of six C-terminal residues of one molecule into the active site of another molecule. The side-chain amide of N14 was hydrogen bonded to the carbonyl of the last peptide bond formed between residues A196 and E197, and also to A71. The CSP profile of mutation was similar to that observed for the N14D mutant. M71A mutation lowered the thermal stability of the protein. CONCLUSION: Our results indicate that the interactions of M71 with N14 and H24 play an important role in optimal positioning of their side-chains relative to the peptidyl-tRNA substrate. Overall, these interactions of M71 are important for the activity, stability, and compactness of the protein. SIGNIFICANCE: The work presented provides original and new structural and dynamics information that significantly enhances our understanding of the network of interactions that govern this enzyme's activity and selectivity.

Structure, dynamics, and biochemical characterization of ADF/cofilin Twinstar from Drosophilamelanogaster.

BACKGROUND: Twinstar is an ADF/cofilin family protein, which is expressed by the tsr gene in Drosophila melanogaster. Twinstar is one of the main regulators of actin cytoskeleton remodelling and is essential for vital cellular processes like cytokinesis and endocytosis. METHODS: We have characterized the structure and dynamics of Twinstar by solution NMR spectroscopy, the interaction of Twinstar with rabbit muscle actin by ITC, and biochemical activities of Twinstar through different biochemical assays using fluorescence spectroscopy and ultra-centrifugation. RESULTS: The solution structure of Twinstar shows characteristic ADF-H fold with well-formed G/F-site and F-site for interaction with actin. The structure possesses an extended F-loop, which is rigid at the base, but flexible towards its apical region. Twinstar shares similar dynamics for the G/F-site with C. elegans homologs, UNC-60A and UNC-60B. However, the dynamics of its F-loop are different from its C. elegans homologs. Twinstar shows strong affinity for ADP-G-Actin and ATP-G-Actin with Kds of ~7.6 nM and ~0.4 µM, respectively. It shows mild F-actin depolymerizing activity and stable interaction with F-actin with a Kd of ~5.0 µM. It inhibits the rate of the nucleotide exchange in a dose dependent manner. CONCLUSION: On the basis of structure, dynamics, and biochemical activity, Twinstar can be taken to execute its biochemical role by facilitating directional growth and maintenance of length of actin filaments. GENERAL SIGNIFICANCE: This study characterizes the structure, backbone dynamics, and biochemical activities of Twinstar of Drosophila, which provides an insight into the regulation of actin dynamics in the member of phylum insecta.

Multistimuli-Responsive Hydrolytically Stable "Smart" Mercury(II) Coordination Polymer.

A new one-dimensional double chain photoluminescent Hg(II) coordination polymer (CP), {[Hg(L)2]·(ClO4)2} n (1), was synthesized using a benzimidazole-appended tripodal tridentate ligand, 1,3,5-tris(benzimidazolylmethyl)benzene (L). The dynamic and flexible framework of 1 allows it to be entitled as first Hg(II)-based CP belonging to the rare category of CPs that exhibit multistimuli-responsive photoluminescence sensing properties and called as "smart" material. The sensitivity of this material via luminescence quenching method showing "turn off" behavior to a range of stimuli, including anions, solvents, and nitroaromatic compounds (NACs), offers more fine-grained control over its properties. 1 can easily adjust its channel dimensions to encapsulate different guest anions forming complete/partial anion-exchanged materials 1A-1B/1C-1E using NO3-, BF4-, OTf-, OTs-, and PF6- anions, respectively. Reversible (1A and 1B) and irreversible (1C-1E) anion exchange behaviors were observed for the complete and partial anion-exchanged products, respectively. The noteworthy feature of the anion-exchanged compounds is their anion-triggered luminescent behavior depending on different properties of anions.The excellent emission in water and high hydrolytic stability of 1 allows its use for rapid and efficient fluorescence-based detections of NACs in aquatic system. The uncoordinated pendant benzimidazole moiety in 1 serves as Lewis basic recognition site for trinitrophenol (TNP) detection, and along with electron- and energy-transfer mechanisms, 1 forms a luminescent probe for detection of TNP with low detection limits (0.55 ppm), exhibiting excellent photostability and recyclability. 1 also represents the first reported Hg(II)-based sensory CP material that can discriminate nitrophenol and nitroaniline isomers through fluorescence sensing.

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv.

BACKGROUND: In the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature. METHODS: We have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques. RESULTS: We have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ. CONCLUSION: Integrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response. GENERAL SIGNIFICANCE: This study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.

Structural characterization of peptidyl-tRNA hydrolase from Mycobacterium smegmatis by NMR spectroscopy.

BACKGROUND: Accumulation of toxic peptidyl-tRNAs in the bacterial cytoplasm is averted by the action of peptidyl-tRNA hydrolase (Pth), which cleaves peptidyl-tRNA into free tRNA and peptide. NMR studies are needed for a protein homolog with a complete crystal structure, for comparison with the NMR structure of Mycobacterium tuberculosis Pth. METHODS: The structure and dynamics of Mycobacterium smegmatis Pth (MsPth) were characterized by NMR spectroscopy and MD simulations. The thermal stability of MsPth was characterized by DSC. RESULTS: MsPth NMR structure has a central mixed seven stranded ß-sheet that is enclosed by six α-helices. NMR relaxation and MD simulations studies show that most of the ordered regions are rigid. Of the substrate binding segments, the gate loop is rigid, the base loop displays slow motions, while the lid loop displays fast timescale motions. MsPth displays high thermal stability characterized by a melting temperature of 61.71°C. CONCLUSION: The NMR structure of MsPth shares the canonical Pth fold with the NMR structure of MtPth. The motional characteristics for the lid region, the tip of helix α3, and the gate region, as indicated by MD simulations and NMR data, are similar for MsPth and MtPth. However, MsPth has relatively less rigid base loop and more compactly packed helices α5 and α6. The packing and the dynamic differences appear to be an important contributing factor to the thermal stability of MsPth, which is significantly higher than that of MtPth. SIGNIFICANCE: MsPth structure consolidates our understanding of the structure and dynamics of bacterial Pth proteins.

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv.

BACKGROUND: We have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv. METHODS: The complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively. RESULTS: Rv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97×10-9M and melting temperature, Tm, of 50.5°C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n=44), in comparison to non-infected healthy individuals (n=20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4. CONCLUSION: Rv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice. GENERAL SIGNIFICANCE: This study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.

Solution structures and dynamics of ADF/cofilins UNC-60A and UNC-60B from Caenorhabditis elegans.

The nematode Caenorhabditis elegans has two ADF (actin-depolymerizing factor)/cofilin isoforms, UNC-60A and UNC-60B, which are expressed by the unc60 gene by alternative splicing. UNC-60A has higher activity to cause net depolymerization, and to inhibit polymerization, than UNC-60B. UNC-60B, on the other hand, shows much stronger severing activity than UNC-60A. To understand the structural basis of their functional differences, we have determined the solution structures of UNC-60A and UNC-60B proteins and characterized their backbone dynamics. Both UNC-60A and UNC-60B show a conserved ADF/cofilin fold. The G-actin (globular actin)-binding regions of the two proteins are structurally and dynamically conserved. Accordingly, UNC-60A and UNC-60B individually bind to rabbit muscle ADP-G-actin with high affinities, with Kd values of 32.25 nM and 8.62 nM respectively. The primary differences between these strong and weak severing proteins were observed in the orientation and dynamics of the F-actin (filamentous actin)-binding loop (F-loop). In the strong severing activity isoform UNC-60B, the orientation of the F-loop was towards the recently identified F-loop-binding region on F-actin, and the F-loop was relatively more flexible with 14 residues showing motions on a nanosecond-picosecond timescale. In contrast, in the weak severing protein isoform UNC-60A, the orientation of the F-loop was away from the F-loop-binding region and inclined towards its own C-terminal and strand ß6. It was also relatively less flexible with only five residues showing motions on a nanosecond-picosecond timescale. These differences in structure and dynamics seem to directly correlate with the differential F-actin site-binding and severing properties of UNC-60A and UNC-60B, and other related ADF/cofilin proteins.

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics.

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide 'SVAP9I' that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4-8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Beyond HbA1c: Identifying Gaps in Glycemic Control Among Children and Young People with Type 1 Diabetes Using Continuous Glucose Monitoring.

OBJECTIVES: To describe continuous glucose monitoring (CGM) derived glycemic variables, and study their association with HbA1c and socio-economic factors in young people with Type 1 diabetes mellitus (T1DM). METHODS: Ninety-two participants [age 15.7 ± 5.0 y (mean ± SD), HbA1c 8.0 ± 1.5% (mean ± SD)] wore a professional CGM sensor for 14 d. RESULTS: Median (IQR) time in range (TIR) was 41 (18)%. Participants spent 41 ± 20% of their day in hyperglycemia (>180 mg/dl), and 14 (13)% in hypoglycemia (<70 mg/dl). High glycemic variability (percent CV >36%) was seen in 92% participants. Older age at diagnosis was associated with higher TIR (ß = 0.267, p = 0.01), lower time above range (TAR) (ß = -0.352, p <0.001), but higher time below range (TBR) (ß = 0.274, p = 0.006). The use of NPH vs. glargine basal insulin was associated with higher TBR (ß = -0.262, p = 0.009) but lower TAR (ß = 0.202, p = 0.041). HbA1c showed negative correlation with TIR (r = -0.449, p <0.001) and TBR (r = -0.466, p <0.001) and positive correlation with TAR (r = 0.580, p <0.001) and mean glucose (r = 0.589, p <0.001). CONCLUSIONS: These data demonstrate wide gaps between the recommended vs. real world glycemic variables in patients with T1DM in this region on multiple daily insulin injections. CGM identifies glycemic variability and complements HbA1c in improving glycemic control.

Basic carbohydrate counting and glycemia in young people with type 1 diabetes in India: A randomized controlled trial.

OBJECTIVES: The aim of this study was to evaluate the effect on glycemic control and acceptability of basic carbohydrate counting (BCC) in children and young adults with type 1 diabetes (T1DM). METHODS: Ninety-two children and young adults (6-25 y of age) with T1DM were randomized to receive either routine nutrition education (RNE), which addressed food groups, glycemic index, and effects of food and exercise on glycemia, or learn BCC with personalized portion size education. A continuous glucose monitoring study and glycosylated hemoglobin (HbA1c) were performed at baseline and after 12 wk. The primary outcome was a change in time-in-range from baseline through 12 wk. A questionnaire on the acceptability of BCC was administered. RESULTS: At 12 wk, there was no significant difference in change in time-in-range between the two groups (BCC group: 1.2 ± 12.2; RNE group: 1.9 ± 12.3; P = 0.786). No significant changes were observed in the percentage of time that blood glucose was >180 or >250 mg/dL; <70 or <54 mg/dL; glycemic variability, percentage of nights with hypoglycemia and HbA1c. In subgroup analysis, there was a significant decrease in HbA1c in the BCC group among participants with higher maternal education (-0.5 versus 0.2, P = 0.042). The total score on the acceptability questionnaire was higher in the BCC group (P = 0.022). CONCLUSION: Among children and young adults in our region with T1DM, BCC provided flexibility in food choices and perception of greater ease of insulin adjustment. Although BCC was equivalent to RNE in terms of glycemic control, larger studies may reveal benefit in outcomes in certain subgroups.

Structural and biophysical characterization of PadR family protein Rv1176c of Mycobacterium tuberculosis H37Rv.

Rv1176c of Mycobacterium tuberculosis H37Rv belongs to the PadR-s1 subfamily of the PadR family of protein. Rv1176c forms a stable dimer in solution. Its stability is characterized by a thermal melting transition temperature (Tm) of 39.4 °C. The crystal structure of Rv1176c was determined at a resolution of 2.94 Å, with two monomers in the asymmetric unit. Each monomer has a characteristic N-terminal winged-helix-turn-helix DNA-binding domain. Rv1176c C-terminal is a coiled-coil dimerization domain formed of α-helices α5 to α7. In the Rv1176c dimer, there is domain-swapping of the C-terminal domain in comparison to other PadR homologs. In the dimer, there is a long inter-subunit tunnel in which different ligands can bind. Rv1176c was found to bind to the promoter region of its own gene with high specificity. M. smegmatis MC2 155 genome lacks homolog of Rv1176c. Therefore, it was used as a surrogate to characterize the functional role of Rv1176c. Expression of Rv1176c in M. smegmatis MC2 155 cells imparted enhanced tolerance towards oxidative stress. Rv1176c expressing M. smegmatis MC2 155 cells exhibited enhanced intracellular survival in J774A.1 murine macrophage cells. Overall, our studies demonstrate Rv1176c to be a PadR-s1 subfamily transcription factor that can moderate the effect of oxidative stress.

Characterization of structure of peptidyl-tRNA hydrolase from Enterococcus faecium and its inhibition by a pyrrolinone compound.

In bacteria, peptidyl-tRNA hydrolase (Pth, E.C. 3.1.1.29) is a ubiquitous and essential enzyme for preventing the accumulation of peptidyl-tRNA and sequestration of tRNA. Pth is an esterase that cleaves the ester bond between peptide and tRNA. Here, we present the crystal structure of Pth from Enterococcus faecium (EfPth) at a resolution of 1.92 Å. The two molecules in the asymmetric unit differ in the orientation of sidechain of N66, a conserved residue of the catalytic site. Enzymatic hydrolysis of substrate α-N-BODIPY-lysyl-tRNALys (BLT) by EfPth was characterized by Michaelis-Menten parameters KM 163.5 nM and Vmax 1.9 nM/s. Compounds having pyrrolinone scaffold were tested for inhibition of Pth and one compound, 1040-C, was found to have IC50 of 180 nM. Antimicrobial activity profiling was done for 1040-C. It exhibited equipotent activity against drug-susceptible and resistant S. aureus (MRSA and VRSA) and Enterococcus (VSE and VRE) with MICs 2-8 µg/mL. 1040-C synergized with gentamicin and the combination was effective against the gentamicin resistant S. aureus strain NRS-119. 1040-C was found to reduce biofilm mass of S. aureus to an extent similar to Vancomycin. In a murine model of infection, 1040-C was able to reduce bacterial load to an extent comparable to Vancomycin.

Highly efficient functionalized MOF-LIC-1 for extraction of U(VI) and Th(IV) from aqueous solution: experimental and theoretical studies.

A set of four new functionalized MOFs, namely MOF-LIC-DPPC, MOF-LIC-GA, MOF-LIC-PCA and MOF-LIC-SA, were synthesized via the post-synthetic modification (PSM) strategy using MOF-LIC-1 for efficient extraction of U(VI) and Th(IV) from an aqueous medium. FTIR, powder XRD, TGA and SEM-EDX were employed for characterization of the functionalized MOFs. Sorption studies for U(VI) and Th(IV) were performed by monitoring the pH and contact time. Interestingly, the modified MOF-LIC-SA displayed rapid (∼5 min) and efficient extraction towards U(VI) and Th(IV) from an aqueous medium and modified MOF-LIC-DPPC displayed enhanced thermal stability (600 °C) compared with the parent MOF-LIC-1 (450 °C). These studies revealed that the grafted functionalities on MOF-LIC-1 possess enhanced sorption efficiency towards U(VI) and Th(IV) as well as thermal stability. MOF-LIC-SA exhibited the highest sorption capacity towards U(VI) and Th(IV), viz. 298 mg g-1 (pH 6) and 149 mg g-1 (pH 6), respectively. Leaching, recyclability, and radiation stability studies were also performed using MOF-LIC-1 MOFs. Additionally, we investigated the nature of U(VI) interactions on MOFs by applying density functional theory (DFT). PSM MOFs with various functionalities display high selectivity and efficient extraction of U(VI) and Th(IV) over a wide pH range (2-9) and also exhibit easy recovery of metal ions from MOFs. These studies reveal that U(VI) and Th(IV) can be extracted from aqueous streams in a pH range from 6 to 8 and potential applications of these MOFs include recovery of U(VI) and Th(IV) from mine water, sea water, etc. The studies reported in the present work also have extensive potential applications for environmental concerns as well as in the nuclear industry.

Solution structure and dynamics of ADF from Toxoplasma gondii.

Toxoplasma gondii ADF (TgADF) belongs to a functional subtype characterized by strong G-actin sequestering activity and low F-actin severing activity. Among the characterized ADF/cofilin proteins, TgADF has the shortest length and is missing a C-terminal helix implicated in F-actin binding. In order to understand its characteristic properties, we have determined the solution structure of TgADF and studied its backbone dynamics from ¹5N-relaxation measurements. TgADF has conserved ADF/cofilin fold consisting of a central mixed ß-sheet comprised of six ß-strands that are partially surrounded by three α-helices and a C-terminal helical turn. The high G-actin sequestering activity of TgADF relies on highly structurally and dynamically optimized interactions between G-actin and G-actin binding surface of TgADF. The equilibrium dissociation constant for TgADF and rabbit muscle G-actin was 23.81 nM, as measured by ITC, which reflects very strong affinity of TgADF and G-actin interactions. The F-actin binding site of TgADF is partially formed, with a shortened F-loop that does not project out of the ellipsoid structure and a C-terminal helical turn in place of the C-terminal helix α4. Yet, it is more rigid than the F-actin binding site of Leishmania donovani cofilin. Experimental observations and structural features do not support the interaction of PIP2 with TgADF, and PIP2 does not affect the interaction of TgADF with G-actin. Overall, this study suggests that conformational flexibility of G-actin binding sites enhances the affinity of TgADF for G-actin, while conformational rigidity of F-actin binding sites of conventional ADF/cofilins is necessary for stable binding to F-actin.

Structural and functional characterization of peptidyl-tRNA hydrolase from Klebsiella pneumoniae.

Klebsiella pneumoniae is a member of the ESKAPE panel of pathogens that are top priority to tackle AMR. Bacterial peptidyl tRNA hydrolase (Pth), an essential, ubiquitous enzyme, hydrolyzes the peptidyl-tRNAs that accumulate in the cytoplasm because of premature termination of translation. Pth cleaves the ester bond between 2' or 3' hydroxyl of the ribose in the tRNA and C-terminal carboxylate of the peptide, thereby making free tRNA available for repeated cycles of protein synthesis and preventing cell death by alleviating tRNA starvation. Pth structures have been determined in peptide-bound or peptide-free states. In peptide-bound state, highly conserved residues F67, N69 and N115 adopt a conformation that is conducive to their interaction with peptide moiety of the substrate. While, in peptide-free state, these residues move away from the catalytic center, perhaps, in order to facilitate release of hydrolysed peptide. Here, we present a novel X-ray crystal structure of Pth from Klebsiella pneumoniae (KpPth), at 1.89 Å resolution, in which out of the two molecules in the asymmetric unit, one reflects the peptide-bound while the other reflects peptide-free conformation of the conserved catalytic site residues. Each molecule of the protein has canonical structure with seven stranded ß-sheet structure surrounded by six α-helices. MD simulations indicate that both the forms converge over 500 ns simulation to structures with wider opening of the crevice at peptide-binding end. In solution, KpPth is monomeric and its 2D-HSQC spectrum displays a single set of well dispersed peaks. Further, KpPth was demonstrated to be enzymatically active on BODIPY-Lys-tRNALys3.