Mutant Complexes Research Articles

Development of New Assays for the Simultaneous Measurement of DNA Double‐Strand Break Repair by Multiple Pathways

Cellular DNA is damaged by both endogenous and exogenous sources. Among the different types of DNA lesions that are formed, double‐strand breaks (DSBs) are considered to be the most consequential lesions. They can cause genomic instability and cellular death if not repaired via one of two pathways: nonhomologous end‐joining (NHEJ) or homologous recombination (HR). Deficiency of DSB repair is implicated in several congenital diseases and cancer‐prone disorders. In the budding yeast Saccharomyces cerevisiae NHEJ is accomplished by the actions of the Yku, Mrx, and Dnl4 protein complexes. Recent experiments in this lab have demonstrated that, in contrast to Yku and Dnl4, the Mrx complex is required for NHEJ repair of some DSB end structures but not others. The major goal of the current project is to characterize the role of Mrx and other protein complexes in repair by NHEJ and HR using newly developed plasmid and PCR fragment‐based assays. Experiments performed for the project demonstrated that repair of DSBs with 3′ single‐stranded DNA overhangs was reduced in yku70, mre11 and dnl4 mutants; however, the defect in NHEJ was strongly suppressed when EXO1, encoding a major 5′‐to‐3′ exonuclease, was inactivated in mre11 cells, i.e., in mre11 exo1 double mutants. By contrast, absence of the Sgs1‐Dna2 nuclease‐helicase complex (in mre11 sgs1 mutants) had no effect. NHEJ repair of DSBs with 5′ single‐stranded DNA overhangs was not changed by inactivation of EXO1 or SGS1. These findings implicate Mrx as a potential protector of DSB ends with 3′ overhangs during NHEJ repair and Exo1 as the main degradative nuclease. Additionally, we have developed new plasmid‐ and PCR fragment‐based repair assays that can analyze NHEJ and HR simultaneously using auxotrophic markers and type IIS restriction endonucleases. We anticipate that the new methods for simultaneous measurement will permit exploration of pathway choice in ways not previously possible.

A molecular docking and dynamics study to screen phytochemicals that target mutant thymidine phosphorylase for colon cancer therapy

Thymidine phosphorylase (TYPH) is an enzyme involved in pyrimidine catabolism and its mutation is associated with chemoresistance of colon cancer to 5-fluorouracil (5-FU) treatment. Here we have analysed the most destabilized mutation of TYPH protein at glycosyltransferase domain where isoleucine alter to alanine at position 214(I214A) that linked to the onset of colon cancer. This study aims to conduct virtual screening of phytochemicals to find novel compounds against mutated TYPH protein. The in silico study aimed to predict the physicochemical properties of phytochemicals and their binding performance with mutated TYPH compared to 5-FU. From the screened phytochemicals, berbamine showed the best binding affinity (−7.2 kcal/mol) with mutant TYPH protein as compared to 5-FU. For further confirmations, the dynamics properties of native, mutant, and docked complex of I214A mutant with berbamine systems were studied through molecular dynamics simulations with a trajectory of 100ns. From root mean square fluctuation, radius of gyration, number of hydrogen bonds, principal component analysis, and free energy landscape, we predicted that the I214A mutant lost its compactness, however, on complex formation with berbamine it gained its compactness. MM/PBSA and molecular docking studies confirmed that berbamine could show potential inhibitory effects against the mutant model of TYPH. Our finding may open the door for its experimental validation and may take as a potential therapeutic against colon cancer treatment in near future.

Molecular dynamic assisted investigation on impact of mutations in deazaflavin dependent nitroreductase against pretomanid: a computational study

In the past decade, TB drugs belonging to the nitroimidazole class, pretomanid and delamanid, have been authorised to treat MDR-TB and XDR-TB. With a novel inhibition mechanism and a reduction in the span of treatment, it is now being administered in various combinations. This approach is not the ultimate remedy since the target protein Deazaflavin dependent nitroreductase (Ddn) has a high mutation frequency, and already pretomanid resistant clinical isolates are reported in various studies. Ddn is essential for M.tuberculosis to emerge from hypoxia, and point mutations in critical residues confer resistance to Nitro-imidazoles. Among the pool of available mutants, we have selected seven mutants viz DdnL49P, DdnY65S, DdnS78Y, DdnK79Q, DdnW88R, DdnY133C, and DdnY136S, all of which exhibited resistance to pretomanid. To address this issue, through computational study primarily by MD simulation, we attempted to elucidate these point mutations’ impact and investigate the resistance mechanism. Hence, the DdnWT and mutant (MT) complexes were subjected to all-atom molecular dynamics (MD) simulations for 100 ns. Interestingly, we observed the escalation of the distance between cofactor and ligand in some mutants, along with a significant change in ligand conformation relative to the DdnWT. Moreover, we confirmed that mutations rendered ligand instability and were ejected from the binding pocket as a result. In conclusion, the results obtained provide a new structural insight and vital clues for designing novel inhibitors to combat nitroimidazole resistance Communicated by Ramaswamy H. Sarma

NADP+ supply adjusts the synthesis of photosystem I in Arabidopsis chloroplasts.

In oxygenic photosynthesis, NADP+ acts as the final acceptor of the photosynthetic electron transport chain and receives electrons via the thylakoid membrane complex photosystem I (PSI) to synthesize NAPDH by the enzyme ferredoxin:NADP+ oxidoreductase. The NADP+/NADPH redox couple is essential for cellular metabolism and redox homeostasis. However, how the homeostasis of these two dinucleotides is integrated into chloroplast biogenesis remains largely unknown. Here, we demonstrate the important role of NADP+ supply for the biogenesis of PSI by examining the nad kinase 2 (nadk2) mutant in Arabidopsis (Arabidopsis thaliana), which demonstrates disrupted synthesis of NADP+ from NAD+ in chloroplasts. Although the nadk2 mutant is highly sensitive to light, the reaction center of photosystem II (PSII) is only mildly and likely only secondarily affected compared to the wild-type. Our studies revealed that the primary limitation of photosynthetic electron transport, even at low light intensities, occurs at PSI rather than at PSII in the nadk2 mutant. Remarkably, this primarily impairs the de novo synthesis of the two PSI core subunits PsaA and PsaB, leading to the deficiency of the PSI complex in the nadk2 mutant. This study reveals an unexpected molecular link between NADK activity and mRNA translation of psaA/B in chloroplasts that may mediate a feedback mechanism to adjust de novo biosynthesis of the PSI complex in response to a variable NADPH demand. This adjustment may be important to protect PSI from photoinhibition under conditions that favor acceptor side limitation.

Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation

Medulloblastoma is the most common malignant brain tumour in children. Genomic studies have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3/4 medulloblastoma. Critically, group 3/4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver. Delineating the role of KBTBD4 mutations thus offers significant opportunities to understand tumour pathogenesis and to exploit the underpinning mechanisms therapeutically. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutation diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in groups 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establish a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identify both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

Characterization of components of a reducing system for SoxR in the cytoplasmic membrane of Escherichia coli.

A reducing system of SoxR, a regulator of redox-active molecules, was identified as rsxABCDGE gene products and RseC in Escherichia coli through genetic studies. We found that ApbE was an additional component of the reducer system. Bacterial two hybrid analysis revealed that these proteins indeed had multiple interactions among themselves. RseC and RsxB formed the core of the complex, interacting with more than five other components. RsxC, the only cytoplasmic component of the system, interacted with SoxR. It might be linked with the rest of the complex via RsxB. Membrane fractions containing the wild type complex but not the mutant complex reduced purified SoxR using NADH as an electron source. These results suggest that Rsx genes, RseC, and ApbE can form a complex using NAD(P)H to reduce SoxR.

Effects of the G48M mutant on the dynamics properties and binding mechanism of PR with SQV and ATV

ABSTRACT The resistance of HIV-1 protease (PR) mutations seriously affects the efficiency of current drugs in the clinical treatment of AIDS. Therefore, it is important to understand the resistance mechanism of mutated residues for the development of PR inhibitors. In this work, the mechanism of G48M mutant PR to inhibitors Saquinavir and Atazanavir was studied by using molecular dynamics simulation combined with free energy prediction. The analysis results showed that, compared with the WT PR, the distance of the flap tip from the protease catalytic binding site and the dihedral angles in the flap regions in the mutant type complexes was changed. These changes resulted in a significant reduction in the occupancy of most of the hydrogen bonds in the mutant complex and the interaction energy of key residues with the inhibitor, which is the main reason for the resistance of the mutation to these inhibitors. This study can provide important guidance for the design of effective inhibitors against HIV-1 PR.

Epigenetic mechanisms affect the curled leaf phenotype in the hypomethylated ddc mutant of Arabidopsis thaliana

The ddc mutant of Arabidopsis thaliana is characterized by pleiotropic phenotypic alterations including a curl-shaped leaf, previously explained by disturbed auxin metabolism and transport. The present study was aimed at further explore the molecular bases underlying the abnormal phenotype of the ddc leaf. We demonstrated that genes specifically related to leaf fate commitment and morphogenesis were misexpressed on developing ddc leaves, such as upregulation of CURLY LEAF (CLF) and downregulation of ASYMMETRIC LEAVES2 (AS2), KNOTTED-like gene from A. thaliana (KNAT6), TEOSINTE-LIKE1 CYCLOIDEA and PROLIFERATING CELL FACTOR 2 (TCP2) and others. The CLF gene, encoding a component of Polycomb repressive complex 2 (PRC2) which adds trimethylation marks at Lys27 of histone H3, was overexpressed in the ddc mutant and concomitantly was correlated with DNA methylation-dependent repression of its negative regulator UCL1. KNAT6, encoding a class 1 KNOX homeotic gene, had increased H3K27me3 trimethylation levels, suggesting it is a target gene of the CLF containing PRC2 complex in the ddc mutant. We postulate that different epigenetic mechanisms modulate expression of genes related to auxin pathways as well as gene targets of Polycomb repressive action, during leaf morphogenesis.

Intermolecular Mechanism and Dynamic Investigation of Avian Influenza H7N9 Virus' Susceptibility to E119V-Substituted Peramivir-Neuraminidase Complex.

The H7N9 virus attaches itself to the human cell receptor protein containing the polysaccharide that terminates with sialic acid. The mutation of neuraminidase at residue E119 has been explored experimentally. However, there is no adequate information on the substitution with E119V in peramivir at the intermolecular level. Therefore, a good knowledge of the interatomic interactions is a prerequisite in understanding its transmission mode and subsequent effective inhibitions of the sialic acid receptor cleavage by neuraminidase. Herein, we investigated the mechanism and dynamism on the susceptibility of the E119V mutation on the peramivir–neuraminidase complex relative to the wildtype complex at the intermolecular level. This study aims to investigate the impact of the 119V substitution on the neuraminidase–peramivir complex and unveil the residues responsible for the complex conformations. We employed molecular dynamic (MD) simulations and extensive post-MD analyses in the study. These extensive computational investigations were carried out on the wildtype and the E119V mutant complex of the protein for holistic insights in unveiling the effects of this mutation on the binding affinity and the conformational terrain of peramivir–neuraminidase E119V mutation. The calculated total binding energy (ΔGbind) for the peramivir wildtype is −49.09 ± 0.13 kcal/mol, while the E119V mutant is −58.55 ± 0.15 kcal/mol. The increase in binding energy (9.46 kcal/mol) is consistent with other post-MD analyses results, confirming that E119V substitution confers a higher degree of stability on the protein complex. This study promises to proffer contributory insight and additional knowledge that would enhance future drug designs and help in the fight targeted at controlling the avian influenza H7N9 virus. Therefore, we suggest that experimentalists collaborate with computational chemists for all investigations of this topic, as we have done in our previous studies.

Impaired nuclear transport induced by juvenile ALS causing P525L mutation in NLS domain of FUS: A molecular mechanistic study

Amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD) are progressive neurological disorders affecting motor neurons. Cellular aggregates of fused in sarcoma (FUS) protein are found in cytoplasm of ALS and FTLD patients. Nuclear localisation signal (NLS) domain of FUS binds to Karyopherin β2 (Kapβ2), which drives nuclear transport of FUS from cytoplasm. Several pathogenic mutations are reported in FUS NLS, which are associated with its impaired nuclear transport and cytoplasmic mis-localisation. P525L mutation in NLS is most commonly found in cases of juvenile ALS (jALS), which affects individuals below 25 years of age. jALS progresses aggressively causing death within a year of its onset. This study elucidates the molecular mechanism behind jALS-causing P525L mutation hindering nuclear transport of FUS. We perform multiple molecular dynamics simulations in aqueous and hydrophobic solvent to understand the effect of the mutation at molecular level. Dynamics of Kapβ2-FUS complex is better captured in hydrophobic solvent compared to aqueous solvent. P525 and Y526 (PY-motif) of NLS exhibit fine-tuned stereochemical arrangement, which is essential for optimum Kapβ2 binding. P525L causes loss of several native contacts at interface leading to weaker binding, which promotes self-aggregation of FUS in cytoplasm. Native complex samples closed conformation, while mutant complex exhibits open conformation exposing hydrophilic residues of Kapβ2 to hydrophobic solvent. Mutant complex also fails to exhibit spring-like motion essential for its transport through nuclear pore complex. This study provides a mechanistic insight of binding affinity between NLS and Kapβ2 that inhibits self-aggregation of FUS preventing the disease condition.

X-ray structure of the Rhodobacter sphaeroides reaction center with an M197 Phe→His substitution clarifies the properties of the mutant complex.

The first steps of the global process of photosynthesis take place in specialized membrane pigment-protein complexes called photosynthetic reaction centers (RCs). The RC of the photosynthetic purple bacterium Rhodobacter sphaeroides, a relatively simple analog of the more complexly organized photosystem II in plants, algae and cyanobacteria, serves as a convenient model for studying pigment-protein interactions that affect photochemical processes. In bacterial RCs the bacteriochlorophyll (BChl) dimer P serves as the primary electron donor, and its redox potential is a critical factor in the efficient functioning of the RC. It has previously been shown that the replacement of Phe M197 by His strongly affects the oxidation potential of P (E m P/P+), increasing its value by 125 mV, as well as increasing the thermal stability of RC and its stability in response to external pressure. The crystal structures of F(M197)H RC at high resolution obtained using various techniques presented in this report clarify the optical and electrochemical properties of the primary electron donor and the increased resistance of the mutant complex to denaturation. The electron-density maps are consistent with the donation of a hydrogen bond from the imidazole group of His M197 to the C2-acetyl carbonyl group of BChl PB. The formation of this hydrogen bond leads to a considerable out-of-plane rotation of the acetyl carbonyl group and results in a 1.2 Å shift of the O atom of this group relative to the wild-type structure. Besides, the distance between BChl PA and PB in the area of pyrrole ring I was found to be increased by up to 0.17 Å. These structural changes are discussed in association with the spectral properties of BChl dimer P. The electron-density maps strongly suggest that the imidazole group of His M197 accepts another hydrogen bond from the nearest water molecule, which in turn appears to form two more hydrogen bonds to Asn M195 and Asp L155. As a result of the F(M197)H mutation, BChl PB finds itself connected to the extensive hydrogen-bonding network that pre-existed in wild-type RC. Dissimilarities in the two hydrogen-bonding networks near the M197 and L168 sites may account for the different changes of the E m P/P+ in F(M197)H and H(L168)F RCs. The involvement of His M197 in the hydrogen-bonding network also appears to be related to stabilization of the F(M197)H RC structure. Analysis of the experimental data presented here and of the data available in the literature points to the fact that the hydrogen-bonding networks in the vicinity of BChl dimer P may play an important role in fine-tuning the redox properties of the primary electron donor.

Mutations in the receptor-binding domain of human SARS CoV-2 spike protein increases its affinity to bind human ACE-2 receptor

The severe acute respiratory syndrome virus-2 (SARS CoV-2) infection has resulted in the current global pandemic. The binding of SARS CoV-2 spike protein receptor-binding domain (RBD) to the human angiotensin converting enzyme-2 (ACE-2) receptor causes the host infection. The spike protein has undergone several mutations with reference to the initial strain isolated during December 2019 from Wuhan, China. A number of these mutant strains have been reported as variants of concern and as variants being monitored. Some of these mutants are known to be responsible for increased transmissibility of the virus. The reason for the increased transmissibility caused by the point mutations can be understood by studying the structural implications and inter-molecular interactions in the binding of viral spike protein RBD and human ACE-2. Here, we use the crystal structure of the RBD in complex with ACE-2 available in the public domain and analyse the 250 ns molecular dynamics (MD) simulations of wild-type and mutants; K417N, K417T, N440K, N501Y, L452R, T478K, E484K and S494P. The ionic, hydrophobic and hydrogen bond interactions, amino acid residue flexibility, binding energies and structural variations are characterized. The MD simulations provide clues to the molecular mechanisms of ACE-2 receptor binding in wild-type and mutant complexes. The mutant spike proteins RBD were associated with greater binding affinity with ACE-2 receptor. Communicated by Ramaswamy H. Sarma

A loop extrusion-independent mechanism contributes to condensin I-mediated chromosome shaping.

Condensin I is a five-subunit protein complex that is central to mitotic chromosome assembly in eukaryotic cells. Despite recent progress, its molecular mechanisms of action remain to be fully elucidated. By using Xenopus egg extracts as a functional assay, we find that condensin I complexes harboring mutations in its kleisin subunit CAP-H produce chromosomes with confined axes in the presence of topoisomerase IIα (topo IIα) and highly compact structures (termed "beans") with condensin-positive central cores in its absence. The bean phenotype depends on the SMC ATPase cycle and can be reversed by subsequent addition of topo IIα. The HEAT repeat subunit CAP-D2, but not CAP-G, is essential for the bean formation. Notably, loop extrusion activities of the mutant complexes cannot explain the chromosomal defects they exhibit in Xenopus egg extracts, implying that a loop extrusion-independent mechanism contributes to condensin I-mediated chromosome assembly and shaping. We provide evidence that condensin-condensin interactions underlie these processes.

The role of the electron-transfer flavoprotein: ubiquinone oxidoreductase following carbohydrate starvation in Arabidopsis cell cultures.

The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.

POT1-TPP1 binding stabilizes POT1, promoting efficient telomere maintenance

Telomeric POT1-TPP1 binding is critical to telomere maintenance and disruption of this complex may lead to cancer. Current data suggests a reduction of intracellular POT1 levels in the absence of TPP1. Here we provide evidence of POT1 plasticity that contributes to its lack of stability in the absence of TPP1 binding. Structural data reveals inter- and intramolecular POT1C domain flexibility in the absence of TPP1. Thermostability and proteolytic resistance assays show that POT1C and the mutant complex POT1C(Q623H)-TPP1(PBD) are less stable than the wild type POT1C-TPP1(PBD), suggesting that TPP1 binding to POT1 stabilizes POT1C and makes it less accessible to proteasomal degradation in the cell. Disruption of the POT1-TPP1 complex such as through cancer-associated mutations leads to a reduction of intracellular POT1, telomere uncapping, and telomere associated DNA damage response (DDR). DDR in turn leads to senescence or genomic instability and oncogenesis.

Effect of mutation of NS2B cofactor residues on Dengue 2 NS2B-NS3 protease complex – an insight to viral replication

Dengue fever is an endemic virus-borne disease that causes many severe ailments, including dengue hemorrhagic fever and dengue shock syndrome. NS2B-NS3 protease is present in all four strains of the dengue virus. NS2B-NS3 is a non-structural protein that performs three distinct functions: protease activity, helicase activity, and nucleoside triphosphatase activity. NS2B-NS3 pro-complex plays a crucial role in viral replication, and NS2B interacts with NS3 protease at a flat active site with an amino acid of the N-terminal region. NS2B acts as a cofactor for NS3 protease. In the current study, the conserved residues of NS2B were identified. Dengue virus-2 NS2B was mutated at the identified conserved amino acid region to investigate the role of NS2B on activation of NS3 pro. Molecular dynamics simulations were performed to investigate the mutated complex’s changes in stability, conformation, and free energy. The EAG mutant complex exhibited more unstable conformation, less hydrogen bond formation, and high binding energy than wild type. This result suggests a vital role of E63, A65, G69 mutation in NS2B for the interruption of activation of the NS3. Communicated by Ramaswamy H. Sarma

Structural Analysis on the Severe Acute Respiratory Syndrome Coronavirus 2 Non-structural Protein 13 Mutants Revealed Altered Bonding Network With TANK Binding Kinase 1 to Evade Host Immune System.

Mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have made this virus more infectious. Previous studies have confirmed that non-structural protein 13 (NSP13) plays an important role in immune evasion by physically interacting with TANK binding kinase 1 (TBK1) to inhibit IFNβ production. Mutations have been reported in NSP13; hence, in the current study, biophysical and structural modeling methodologies were adapted to dissect the influence of major mutations in NSP13, i.e., P77L, Q88H, D260Y, E341D, and M429I, on its binding to the TBK1 and to escape the human immune system. The results revealed that these mutations significantly affected the binding of NSP13 and TBK1 by altering the hydrogen bonding network and dynamic structural features. The stability, flexibility, and compactness of these mutants displayed different dynamic features, which are the basis for immune evasion. Moreover, the binding was further validated using the MM/GBSA approach, revealing that these mutations have higher binding energies than the wild-type (WT) NSP13 protein. These findings thus justify the basis of stronger interactions and evasion for these NSP13 mutants. In conclusion, the current findings explored the key features of the NSP13 WT and its mutant complexes, which can be used to design structure-based inhibitors against the SARS-CoV-2 new variants to rescue the host immune system.

Computational investigations of structural and interactive patterns of human MTHFR wild and mutant type under different physiological conditions: An insight into underlying perturbations.

Methylene Tetrahydrofolate Reductase (MTHFR), involved in regulation of methionine and folate synthesis is a potential marker of Alzheimer's Disease (AD). Metabolic folate pathways along with methyl donor reactions are crucial in AD pathogenesis. A222V mutation denatures MTHFR due to thermolability and deranges methionine and folate levels resulting in increased plasma homocysteine levels. Also, there occurs increased GSK3β and decreased Protein Phosphatase 2A levels which are anticipated to be involved in phosphorylation of Amyloid β and Tau proteins. In this study, structural and interactive patterns of wild type and mutated (A222V) MTHFR with the substrate FAD were studied. X-ray crystallographic structure of MTHFR (PDB ID: 6CFX) was retrieved from PDB. The mutant protein was created by mutating A to V at 222nd position. The models were validated through ERRAT, Verify 3D and PROCHECK servers. Structure of FAD was retrieved and redocked at the same positions of created homodimer and Prime MM-GBSA was calculated for both wild and mutant protein complexes. The ligand behaviour at different pH and temperatures were studied using Desmond and GROMACS respectively. The stability and interactive patterns of the complexes were studied through Molecular Dynamics. Protein and ligand RMSD, RMSF, Radius of Gyration (RoG), Solvent accessible surface, secondary structure changes and MM-PBSA were examined. The created homodimers were found to be stable and around 89% of the residues were found to be present in favoured regions of Ramachandran plot. ERRAT and Verify 3D plots yielded best results. No much variations in docking scores of FAD ligands in both chains of the proteins were observed; whereas slight variations were seen in the MM-GBSA and MM-PBSA energies of the ligands. FAD in chain A was found to possess high energies rather than FAD in chain B in both proteins. RMSD, RMSF and RoG were found to be stable throughout the simulations. The behaviour of FAD was found to be different in mutated type at different temperatures. Computational studies offer better insights into the structural changes in proteins and their relationship with disease conditions.

Structures of the HER2-HER3-NRG1β complex reveal a dynamic dimer interface.

Human epidermal growth factor receptor 2 (HER2) and HER3 form a potent pro-oncogenic heterocomplex1-3 upon binding of growth factor neuregulin-1β (NRG1β). The mechanism by which HER2 and HER3 interact remains unknown in the absence of any structures of the complex. Here we isolated the NRG1β-bound near full-length HER2-HER3 dimer and, using cryo-electron microscopy, reconstructed the extracellulardomain module, revealing unexpected dynamics at the HER2-HER3 dimerization interface. We show that the dimerization arm of NRG1β-bound HER3 is unresolved because the apo HER2 monomer does not undergo a ligand-induced conformational change needed to establish a HER3 dimerization arm-binding pocket. In a structure of the oncogenic extracellular domain mutant HER2(S310F), we observe a compensatory interaction with the HER3 dimerization arm that stabilizes the dimerization interface. Both HER2-HER3 and HER2(S310F)-HER3 retain the capacity to bind to the HER2-directed therapeutic antibody trastuzumab, but the mutant complex does not bind to pertuzumab. Our structure of the HER2(S310F)-HER3-NRG1β-trastuzumab Fab complex reveals that the receptor dimer undergoes a conformational change to accommodate trastuzumab. Thus, similar to oncogenic mutations, therapeutic agents exploit the intrinsic dynamics of the HER2-HER3 heterodimer. The unique features of a singly liganded HER2-HER3 heterodimer underscore the allosteric sensing of ligand occupancy by the dimerization interface and explain why extracellular domains of HER2 do not homo-associate via a canonical active dimer interface.

Entospletinib (ENTO) and Decitabine (DEC) Combination Therapy in Older Newly Diagnosed (ND) Acute Myeloid Leukemia (AML) Patients with Mutant TP53 or Complex Karyotype Is Associated with Poor Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial

Entospletinib (ENTO) and Decitabine (DEC) Combination Therapy in Older Newly Diagnosed (ND) Acute Myeloid Leukemia (AML) Patients with Mutant TP53 or Complex Karyotype Is Associated with Poor Response and Survival: A Phase 2 Sub-Study of the Beat AML Master Trial