Helix H3 Research Articles

In vivo emergence of resistance to ceftazidime/avibactam through modification of chromosomal AmpC β-lactamase in Klebsiella aerogenes.

We describe the in vivo emergence of resistance to ceftazidime/avibactam via modification of AmpC in a clinical Klebsiella aerogenes isolate during therapy with this combination. Paired ceftazidime/avibactam-susceptible/resistant isolates were obtained before and during ceftazidime/avibactam treatment. Whole genome sequencing revealed a differential mutation in AmpC (R148W) in the ceftazidime/avibactam-resistant isolate. Molecular cloning and structural studies confirmed the impact of this substitution, which affects the architecture of the H10 helix, on the evolved resistant phenotype.

Aquaporin channels in desalination: Mechanical properties and operational load analysis

The scarcity of freshwater sources and the global demand for drinking water have spurred researchers worldwide to develop new and efficient desalination and water purification technologies. One promising method is desalination using aquaporin (AQP) channels, which are highly regarded for their biocompatibility and exceptional desalination efficiency. However, there is limited information on the mechanical behavior of these proteins under operational loads and how they maintain their function and resilience over time. This research employs all-atom molecular dynamics simulation to calculate the mechanical properties of the channels at the nanoscale. It also uses the finite element method to analyze the behavior of channels in vesicles embedded in composite plates at the macroscale under operational loads and conditions. Our research shows that the force on vesicle walls changes considerably with applied pressure, peaking at 14 pN at 55 bar. This variability highlights the need to carefully assess the weakest parts of the nanochannels, especially the helices HB and H1, which are susceptible to high strain and possible unfolding under extended stress. The results indicate the force tolerance threshold of the subsystems, guiding the application of appropriate force conditions for optimal performance and long-term system maintenance. Beyond desalination systems, the findings offer useful information for researchers working with aquaporin nanochannels in applications such as targeted drug release systems based on protein nanovalves, solid-state sequencing systems, and more.

Insights into lipid-modified recognition of Apolipoprotein E3 to extra-cellular domain of TREM2 associated with Alzheimer’s disease

E3 genotype of apolipoprotein E (ApoE) and triggering receptor expressed on myeloid cells 2 (TREM2) are critical genetic risk factors for late-onset Alzheimer’s diseases (AD) that influences the formation of plaques and neurofibrillary tangles associated with AD pathogenesis. In addition, variation in genetic association of ApoE and TREM2 in presence of lipids have been advocated to potentially regulate different aspects of AD pathology by facilitating the clearance of amyloid beta (Aβ) from brain. However, the proteinopathy and functional mechanism underlying their molecular interaction in presence of lipid remains poorly understood. Herein, to shed inherent insights into the mode of ApoE3-TREM2 interaction in both lipid and aqueous medium, we have used a combination of different state-of-the-art computational tools including knowledge-based protein–protein docking and molecular dynamics (MD) simulations. Remarkably, we find the direct involvement of regulatory complementarity-determining region-2 (CDR2) loop of TREM2 in interaction with the major hinge region and helix H4 of ApoE3. We further anticipate that ApoE3 attains distinct conformational states and forms an open and extended cavity-like structure in ApoE3-TREM2 complex in presence of lipids. Our data also exhibits reduced hydrophobicity in the ApoE3 binding interface of TREM2, which sets a baseline for reduced binding affinity towards ApoE3. Therefore, we hypothesize that this decline in hydrophobic index might have resulted due to structural variations in the H4 helix and major hinge region leading to altered TREM2 conformation in presence of lipids. Altogether, our findings demonstrate the effect of phospholipids on the structural dynamics and conformation of ApoE3, which contributes to our understanding of how APOE and TREM2 influences the development of AD. However, further experimental studies are necessary to validate and explore our findings which will open up new avenues towards development of novel therapeutic strategies for AD.

Impact of transferable β-lactamases and intrinsic AmpC amino acid substitutions on the activity of cefiderocol against wild-type and iron uptake-deficient mutants of Pseudomonas aeruginosa.

We aimed to analyse the interplay between impaired iron uptake and β-lactamases on cefiderocol resistance in Pseudomonas aeruginosa. Thirty-one transferable β-lactamases and 16 intrinsic P. aeruginosa AmpC (PDC) variants were cloned and expressed in wild-type (PAO1) and iron uptake-deficient (PAO ΔpiuC) P. aeruginosa backgrounds. MICs of cefiderocol and antipseudomonal β-lactams were determined by reference broth microdilution. Relative to PAO1, deletion of piuC caused a specific 16-fold decrease in cefiderocol activity but negligible effects on the activity of other β-lactams. Among transferable β-lactamases, SHV-12, KPC Ω-loop mutants, NDMs and OXA-15 showed cefiderocol MIC values above the clinical breakpoint (2 mg/L) when expressed in PAO1. When expressed in PAO ΔpiuC, these and the transformants harbouring PER-1, VEB-1, KPC-2, KPC-3, VIM-1, CMY-2, OXA-2 and OXA-14 showed increased MIC values from 16 to >256 mg/L. The PDC variants carrying the Ω-loop changes ΔP215-G222 (PDC-577), E219K (PDC-221 and PDC-558) and the H10 helix change L293P (PDC-219) had the greatest impact on cefiderocol resistance, with MICs of 2-4 mg/L in PAO1 and of up to 32-64 mg/L in PAO ΔpiuC. Widespread enzymes such as GES, CTX-M-9, CTX-M-15, VIM-2-like enzymes, IMPs, DHA-1, FOX-4, OXA-10, OXA-48 and the other PDC variants tested had weaker effects on cefiderocol resistance. We add evidence about the effect of the interplay between iron uptake and β-lactamases on the acquisition of cefiderocol resistance in P. aeruginosa. These findings may help to anticipate the emergence of resistance and optimize the use of cefiderocol against P. aeruginosa infections.

Charge Relaying within a Phospho-Motif Rescue Binding Competency of a Disordered Transcription Factor.

Structural disorder in proteins is central to cellular signaling, where conformational plasticity equips molecules to promiscuously interact with different partners. By engaging with multiple binding partners via the rearrangement of its three helices, the nuclear coactivator binding domain (NCBD) of the CBP/p300 transcription factor is a paradigmatic example of promiscuity. Recently, molecular simulations and experiments revealed that, through the establishment of long-range electrostatic interactions, intended as salt-bridges formed between the post-translationally inserted phosphate and positively charged residues in helix H3 of NCBD, phosphorylation triggers NCBD compaction, lowering its affinity for binding partners. By means of extensive molecular simulations, we here investigated the effect of short-range electrostatics on the conformational ensemble of NCBD, by monitoring the interactions between a phosphorylated serine and conserved positively charged residues within the NCBD phospho-motif. We found that empowering proximal electrostatic interactions, as opposed to long-range electrostatics, can reshape the NCBD ensemble rescuing the binding competency of phosphorylated NCBD. Given the conservation of positive charges in phospho-motifs, proximal electrostatic interactions might dampen the effects of phosphorylation and act as a relay to regulate phosphorylated intrinsically disordered proteins, ultimately tuning the binding affinity for different cellular partners.

Mutations found in cancer patients compromise DNA binding of the winged helix protein STK19

Serine/threonine protein kinase 19 (STK19) has been reported to phosphorylate and activate oncogenic NRAS to promote melanomagenesis. However, concerns have been raised about whether STK19 is a kinase. STK19 has also been identified as a putative factor involved in the transcription-coupled nucleotide excision repair (TC-NER) pathway. In this study, we determined the 1.32 Å crystal structure of human STK19. The structure reveals that STK19 is a winged helix (WH) protein consisting of three tandem WH domains. STK19 binds more strongly to double-stranded DNA and RNA (dsDNA/dsRNA) than to ssDNA. A positively charged patch centered on helix WH3-H1 contributes to dsDNA binding, which is unusual because the WH domain typically uses helix H3 as the recognition helix. Importantly, mutations of the conserved residues in the basic patch, K186N, R200W, and R215W, are found in cancer patients, and these mutations compromise STK19 DNA binding. Other mutations have been predicted to produce a similar effect, including two mutations that disrupt the nuclear localization signal (NLS) motif. These mutations may indirectly impact the DNA binding capacity of STK19 by interfering with its nuclear localization.

Cloning, Expression, and Bioinformatics Modeling of Human Papillomavirus Type 52 L1/L2 Chimeric Protein in Escherichia coli BL21 (DE3)

Human papillomavirus (HPV) L1 major capsid protein generates a highly immunogenic virus like particles (VLPs), which have been used as the main component of its prophylactic vaccine. However, the neutralizing antibodies against L1 VLPs are mostly type specific and may not be effective to prevent infection from different strains of HPV. On the other hand, HPV L2 minor capsid protein has low antigenic variation, thus can induce cross-neutralization. This study aims to obtain HPV 52 L1/L2 chimeric protein, which is designed based on HPV type 52 as one of the most circulated high-risk types in Indonesia, to develop a broad-spectrum HPV vaccine. Substitution of HPV 52 H4 helix L1 region with an HPV 52 L2 epitope was carried out using overlap extension PCR. HPV 52 L1/L2 chimeric gene was constructed into pET-SUMO expression vector and expressed in Escherichia coli BL21 (DE3). Bioinformatics modeling suggested that L2 epitope was located inside of the loop region in monomer form, and on the contrary, it was located outside of the pentamer surface. Furthermore, B cell and T cell epitopes predictions were conducted using Immune Epitope Database (IEDB) analysis. The B cell epitopes prediction revealed eleven potential epitopes, whereas the T cell epitopes prediction showed seven potential epitopes for each MHC class I and MHC class II. This study showed that HPV 52 L1/L2 chimeric protein has the potential to induce cross-neutralizing antibodies and can be developed as a promising candidate for a new HPV vaccine.

A Combined Molecular Dynamics and Hydropathic INTeraction (HINT) Approach to Investigate Protein Flexibility: The PPARγ Case Study.

Molecular Dynamics (MD) is a computational technique widely used to evaluate a molecular system's thermodynamic properties and conformational behavior over time. In particular, the energy analysis of a protein conformation ensemble produced though MD simulations plays a crucial role in explaining the relationship between protein dynamics and its mechanism of action. In this research work, the HINT (Hydropathic INTeractions) LogP-based scoring function was first used to handle MD trajectories and investigate the molecular basis behind the intricate PPARγ mechanism of activation. The Peroxisome Proliferator-Activated Receptor γ (PPARγ) is an emblematic example of a highly flexible protein due to the extended ω-loop delimiting the active site, and it is responsible for the receptor's ability to bind chemically different compounds. In this work, we focused on the PPARγ complex with Rosiglitazone, a common anti-diabetic compound and analyzed the molecular basis of the flexible ω-loop stabilization effect produced by the Oleic Acid co-binding. The HINT-based analysis of the produced MD trajectories allowed us to account for all of the energetic contributions involved in interconverting between conformational states and describe the intramolecular interactions between the flexible ω-loop and the helix H3 triggered by the allosteric binding mechanism.

3D-QSAR and Molecular Dynamics Study of Isoxazole Derivatives to Identify the Structural Requirements for Farnesoid X Receptor (FXR) Agonists.

The farnesoid X receptor (FXR) has been recognized as a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). FXR agonists benefit NAFLD by modulating bile acid synthesis and transport, lipid metabolism, inflammation, and fibrosis pathways. However, there are still great challenges involved in developing safe and effective FXR agonists. To investigate the critical factors contributing to their activity on the FXR, 3D-QSAR molecular modeling was applied to a series of isoxazole derivatives, using comparative molecular field analysis (CoMFA (q2 = 0.664, r2 = 0.960, r2pred = 0.872)) and comparative molecular similarity indices analysis (CoMSIA (q2 = 0.706, r2 = 0.969, r2pred = 0.866)) models, which demonstrated strong predictive ability in our study. The contour maps generated from molecular modeling showed that the presence of hydrophobicity at the R2 group and electronegativity group at the R3 group in these compounds is crucial to their agonistic activity. A molecular dynamics (MD) simulation was carried out to further understand the binding modes and interactions between the FXR and its agonists in preclinical or clinical studies. The conformational motions of loops L: H1/H2 and L: H5/H6 in FXR-ligand binding domain (LBD) were crucial to the protein stability and agonistic activity of ligands. Hydrophobic interactions were formed between residues (such as LEU287, MET290, ALA291, HIS294, and VAL297) in helix H3 and ligands. In particular, our study found that residue ARG331 participated in salt bridges, and HIS447 participated in salt bridges and hydrogen bonds with ligands; these interactions were significant to protein-ligand binding. Eight new potent FXR agonists were designed according to our results, and their activities were predicted to be better than that of the first synthetic FXR agonist, GW4064.

Screening of the Antagonistic Activity of Potential Bisphenol A Alternatives toward the Androgen Receptor Using Machine Learning and Molecular Dynamics Simulation.

Over the past few decades, extensive research has indicated that exposure to bisphenol A (BPA) increases the health risks in humans. Toxicological studies have demonstrated that BPA can bind to the androgen receptor (AR), resulting in endocrine-disrupting effects. In recent investigations, many alternatives to BPA have been detected in various environmental media as major pollutants. However, related experimental evaluations of BPA alternatives have not been systematically implemented for the assessment of chemical safety and the effects of structural characteristics on the antagonistic activity of the AR. To promote the green development of BPA alternatives, high-throughput toxicological screening is fundamental for prioritizing chemical tests. Therefore, we proposed a hybrid deep learning architecture that combines molecular descriptors and molecular graphs to predict AR antagonistic activity. Compared to previous models, this hybrid architecture can extract substantial chemical information from various molecular representations to improve the model's generalization ability for BPA alternatives. Our predictions suggest that lignin-derivable bisguaiacols, as alternatives to BPA, are likely to be nonantagonist for AR compared to bisphenol analogues. Additionally, molecular dynamics (MD) simulations identified the dihydrotestosterone-bound pocket, rather than the surface, as the major binding site of bisphenol analogues. The conformational changes of key helix H12 from an agonistic to an antagonistic conformation can be evaluated qualitatively by accelerated MD simulations to explain the underlying mechanism. Overall, our computational study is helpful for toxicological screening of BPA alternatives and the design of environmentally friendly BPA alternatives.

Oncogenic R248W mutation induced conformational perturbation of the p53 core domain and the structural protection by proteomimetic amyloid inhibitor ADH-6.

The involvement of p53 aggregation in cancer pathogenesis emphasizes the importance of unraveling the mechanisms underlying mutation-induced p53 destabilization. And understanding how small molecule inhibitors prevent the conversion of p53 into aggregation-primed conformations is pivotal for the development of therapeutics targeting p53-aggregation-associated cancers. A recent experimental study highlights the efficacy of the proteomimetic amyloid inhibitor ADH-6 in stabilizing R248W p53 and inhibiting its aggregation in cancer cells by interacting with the p53 core domain (p53C). However, it remains mostly unclear how R248W mutation induces destabilization of p53C and how ADH-6 stabilizes this p53C mutant and inhibits its aggregation. Herein, we conducted all-atom molecular dynamics simulations of R248W p53C in the absence and presence of ADH-6, as well as that of wild-type (WT) p53C. Our simulations reveal that the R248W mutation results in a shift of helix H2 and β-hairpin S2-S2' towards the mutation site, leading to the destruction of their neighboring β-sheet structure. This further facilitates the formation of a cavity in the hydrophobic core, and reduces the stability of the β-sandwich. Importantly, two crucial aggregation-prone regions (APRs) S9 and S10 are disturbed and more exposed to solvent in R248W p53C, which is conducive to p53C aggregation. Intriguingly, ADH-6 dynamically binds to the mutation site and multiple destabilized regions in R248W p53C, partially inhibiting the shift of helix H2 and β-hairpin S2-S2', thus preventing the disruption of the β-sheets and the formation of the cavity. ADH-6 also reduces the solvent exposure of APRs S9 and S10, which disfavors the aggregation of R248W p53C. Moreover, ADH-6 can preserve the WT-like dynamical network of R248W p53C. Our study elucidates the mechanisms underlying the oncogenic R248W mutation induced p53C destabilization and the structural protection of p53C by ADH-6.

The dynamics of Escherichia coli FtsZ dimer

The E. coli FtsZ dimer was studied to gain insights into FtsZ protofilament formation. In the simulation study of the M. janaschii dimer it was found that the monomer-monomer contacts in the GDP bound dimer is lower which results in the high curvature of the GDP bound protofilaments. In this study, the E. coli FtsZ dimer was simulated. The initial structure was obtained from our previous study in which we had simulated the E. coli FtsZ monomer with its C-terminal IDR (Intrinsically Disordered Region). The M. janaschii FtsZ dimer subunit contacts were used as the starting configuration. Simulations of the dimer were performed with GTP and with GDP. It is found that the central helix H5 closes by about 15 degrees in the simulation with GTP than in the simulation with GDP. The C-terminal IDR and the C-terminal domain region between SC2 and HC2 are found to have much high flexibility and hence exhibit domain motion. Communicated by Ramaswamy H. Sarma

Slc11 Synapomorphy: A Conserved 3D Framework Articulating Carrier Conformation Switch.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal ions (Me2+) such as manganese and iron-vital elements coveted during infection. The Slc11 mechanism of high-affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution, Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outwardly open (OO) to inwardly open (IO) carriers. In silico mutagenesis targeting (i) a set of (evolutionarily coupled) sites that may define Slc11 function (putative synapomorphy) and (ii) residues from networked communities evolving during MCb transition indicates that Slc11 synapomorphy primarily instructs a Me2+-selective conformation switch which unlocks carrier inner gate and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to an altered intramolecular H+ network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to an OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, with extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks the emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying that it constitutes a 3D network of residues articulating a Me2+-selective carrier conformation switch which is maintained in fast-evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

OR11-02 Structural Determinants of Pure Antiestrogenic Activity

Abstract Disclosure: A. Vallet: None. M. Diennet: None. K. Thiombane: None. A. Vivet: None. S. Weber: None. M. El Ezzy: None. F. Shaikh: None. J. Poupart: None. R. Mendoza-Sanchez: None. D. Schuetz: None. A. Marinier: None. G.L. Greene: None. S.W. Fanning: None. S.N. Mader: None. Inhibitory activities of antiestrogens on estrogen receptor alpha (ERα) range from mixed antagonism/agonism (selective ER modulators; SERMs) to complete antiestrogenicity associated with accelerated ERα turnover (selective ER degraders; SERDs). Using a panel of SERMs, SERDs and PROTACs, we show that antiestrogens induce variable degrees of SUMOylation of ERα and that efficient induction of SUMOylation, rather than increased suppression of coactivator recruitment or accelerated ERα degradation, correlates with suppression of ERα basal and induced transcriptional activity in reporter assays. Antiestrogens did not induce SUMOylation of ERβ and did not suppress its basal transcriptional activity in the same assays. Chimeras between the two receptors identified ERα-specific residues required for induction of SUMOylation by antiestrogens and revealed differences according to the antiestrogen structure. In addition, induction of ERα SUMOylation by antiestrogens depended on the hydrophobicity of N-terminal residues of ligand binding domain (LBD) helix H12. L536 mutations, including those occurring in endocrine therapy-resistant breast cancer, abolished both induction of ERα SUMOylation and pure antiestrogenicity. Structures of the L536S human ERα LBD bound to fulvestrant analogs and molecular dynamics simulations revealed dynamic side chain interactions with ERα H12 in the coactivator-binding groove, and predict the impact of SUMOylation-suppressing L536 mutations on these interactions. Together, our results show that antiestrogen-induced ERα SUMOylation is associated with complete suppression of its transcriptional activity and provide insights into the structural determinants of pure antiestrogenicity. Presentation Date: Friday, June 16, 2023

Molecular insights into recognition of GUCY2C by T-cell engaging bispecific antibody anti-GUCY2CxCD3

The intestinal epithelial receptor Guanylyl Cyclase C (GUCY2C) is a tumor-associated cell surface antigen expressed across gastrointestinal malignancies that can serve as an efficacious target for colorectal cancer immunotherapy. Here, we describe a yeast surface-display approach combined with an orthogonal peptide-based mapping strategy to identify the GUCY2C binding epitope of a novel anti-GUCY2CxCD3 bispecific antibody (BsAb) that recently advanced into the clinic for the treatment of cancer. The target epitope was localized to the N-terminal helix H2 of human GUCY2C, which enabled the determination of the crystal structure of the minimal GUCY2C epitope in complex with the anti-GUCY2C antibody domain. To understand if this minimal epitope covers the entire antibody binding region and to investigate the impact of epitope position on the antibody’s activity, we further determined the structure of this interaction in the context of the full-length extracellular domain (ECD) of GUCY2C. We found that this epitope is positioned on the protruding membrane-distal helical region of GUCY2C and that its specific location on the surface of GUCY2C dictates the close spatial proximity of the two antigen arms in a diabody arrangement essential to the tumor killing activity of GUCY2CxCD3 BsAb.

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit.

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA-RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

Alu RNA fold links splicing with signal recognition particle proteins.

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact of successive primate mutations and their combinations on inclusion of a sense-oriented AluJ exon in the human F8 gene. We show that the splicing outcome was better predicted by consecutive RNA conformation changes than by computationally derived splicing regulatory motifs. We also demonstrate an involvement of SRP9/14 (signal recognition particle) heterodimer in splicing regulation of Alu-derived exons. Nucleotide substitutions that accumulated during primate evolution relaxed the conserved left-arm AluJ structure including helix H1 and reduced the capacity of SRP9/14 to stabilize the closed Alu conformation. RNA secondary structure-constrained mutations that promoted open Y-shaped conformations of the Alu made the Alu exon inclusion reliant on DHX9. Finally, we identified additional SRP9/14 sensitive Alu exons and predicted their functional roles in the cell. Together, these results provide unique insights into architectural elements required for sense Alu exonization, identify conserved pre-mRNA structures involved in exon selection and point to a possible chaperone activity of SRP9/14 outside the mammalian signal recognition particle.

Picloram binds to the h1 and h4 helices of HSA domain IIIA at drug binding site 2

Picloram (PC) is a systemic herbicide that controls herbaceous weeds and woody plants. HSA, the most abundant protein in human physiology, binds to all exogenic and endogenic ligands. PC is a stable molecule (t1/2∼157–513 days) and a potential threat to human health via the food chain. HSA and PC binding study has been done to decipher the location and thermodynamics of binding. It has been studied with prediction tools like autodocking and MD simulation and then confirmed with fluorescence spectroscopy. HSA fluorescence was quenched by PC at pH 7.4 (N state), pH 3.5 (F state), and pH 7.4 with 4.5 M urea (I state) at temperatures 283 K, 297 K, and 303 K. The location of binding was found to be interdomain between II and III which overlaps with drug binding site 2. The binding was spontaneous, and entropy-driven that show a noticeable increase in binding with the increase in temperature. No secondary structure change at the native state has been observed due to binding. The binding results are important to understand the physiological assimilation of PC. In silico predictions and the results of spectroscopic studies unambiguously indicate the locus and nature of the binding.

Single-Molecule Monitoring of Membrane Association of the Necroptosis Executioner MLKL with Discernible Anchoring and Insertion Dynamics.

The dynamics of membrane proteins that are well-folded in water and become functional after self-insertion into cell membranes is not well understood. Herein we report on single-molecule monitoring of membrane association dynamics of the necroptosis executioner MLKL. We observed that, upon landing, the N-terminal region (NTR) of MLKL anchors onto the surface with an oblique angle and then is immersed in the membrane. The anchoring end does not insert into the membrane, but the opposite end does. The protein is not static, switching slowly between water-exposed and membrane-embedded conformations. The results suggest a mechanism for the activation and function of MLKL in which exposure of H4 is critical for MLKL to adsorb on the membrane, and the brace helix H6 regulates MLKL rather than inhibits it. Our findings provide deeper insights into membrane association and function regulation of MLKL and would have impacts on biotechnological applications.

Structural insights into the mechanism of resistance to bicalutamide by the clinical mutations in androgen receptor in chemo-treatment resistant prostate cancer

Despite advanced diagnosis and detection technologies, prostate cancer (PCa) is the most prevalent neoplasms in males. Dysregulation of the androgen receptor (AR) is centrally involved in the tumorigenesis of PCa cells. Acquisition of drug resistance due to modifications in AR leads to therapeutic failure and relapse in PCa. An overhaul of comprehensive catalogues of cancer-causing mutations and their juxta positioning on 3D protein can help in guiding the exploration of small drug molecules. Among several well-studied PCa-specific mutations, T877A, T877S and H874Y are the most common substitutions in the ligand-binding domain (LBD) of the AR. In this study, we combined structure as well as dynamics-based in silico approaches to infer the mechanistic effect of amino acid substitutions on the structural stability of LBD. Molecular dynamics simulations allowed us to unveil a possible drug resistance mechanism that acts through structural alteration and changes in the molecular motions of LBD. Our findings suggest that the resistance to bicalutamide is partially due to increased flexibility in the H12 helix, which disturbs the compactness, thereby reducing the affinity for bicalutamide. In conclusion, the current study helps in understanding the structural changes caused by mutations and could assist in the drug development process. Communicated by Ramaswamy H. Sarma