Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

How Dedicated Ribosomes Translate a Leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

Structural insight into translation initiation of the λcl leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fmet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entry channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

Development of antioxidant-rich edible active films and coatings incorporated with de-oiled ethanolic green algae extract: a candidate for prolonging the shelf life of fresh produce.

The concept of sustainability and the substitution of non-biodegradable packaging using biodegradable packaging has attracted gigantic interest. The objective of the present study was to revalorize the biowaste "de-oiled green algae biomass (DAB)" of Dunaliella tertiolecta using a green approach and the development of biodegradable chitosan (CS)-based edible active biocomposite films and coatings for prolonging the shelf life of fresh produce. Ultrasound-assisted green extraction was conducted using food-grade solvent ethanol for obtaining the bio-actives, namely "crude algae ethanolic extract (CAEE)" from DAB. The edible films (CS/CAEE) and coating solutions were developed by incorporating CAEE with varying concentrations (0 to 28%). The CAEE was subjected to MALDI-TOF-MS, NMR, and other biochemical analyses, and was found to be rich in DPPH antioxidant activity (∼40%). The CS/CAEE films were fabricated using a solvent casting method and characterized by several biochemical and physicochemical (FESEM, TGA, FTIR, XRD, WVP, UTM, and rheological) characterization techniques. The addition of CAEE into the CS matrix reduced the maximum film transparency (∼20%), water vapor permeability (∼60%); improved the crystallinity (∼24%), tensile strength (∼25%), and antioxidant activity (∼27%); and exhibited UV-Vis blocking properties as compared to the control film. Besides, the developed coating solutions and CAEE showed biocompatibility with BHK-21 fibroblast cells and antimicrobial activity against common food pathogens. The developed coating solution was applied on green chilli using a dipping method and stored at ambient temperature (25 ± 2 °C, 50-70 % RH) for 10 days. The shelf life of chillies was extended without altering the quality as compared to uncoated green chillies. Therefore, the formulated coating could be applicable for prolonging the shelf life of fresh produce.

PRFF Peptide Mimic Interferes with Toxic Fibrin-Aß42 Interaction by Emulating the Aß Binding Interface on Fibrinogen.

Cerebrovascular dysfunction is a common phenomenon in Alzheimer's patients, where fibrinogen is a major player. With the blood-brain barrier compromised, fibrinogen gains access to the brain, where its interaction with Aß42 results in plasmin-resistant abnormal blood clots that are deposited in the cerebral blood vessels, a condition commonly encountered in Alzheimer's disease (AD) patients called cerebral amyloid angiopathy (CAA). So far, there have been no effective therapeutics available to combat AD-associated CAA. This study reports a 13-amino acid peptide (Pα-NPGRPEPGSAGTW) as a potential inhibitor of the fibrin-Aß42 interaction along with the property to dissolve pre-existing plasmin-resistant abnormal clots. Strikingly, the identified sequence was found to be partially similar to a fragment of the fibrinogen α-chain reported to bind Aß42, the plasmin-resistant fibrinogen fragment (PRFF). Mechanistically, Pα interacts with Aß42 in place of fibrinogen, thus inhibiting the toxic fibrin-Aß42 interaction. However, it does not interfere with normal fibrin polymerization.

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

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

DNA-Bound p53-DNA-Binding Domain Interconverts between Multiple Conformations: Implications for Partner Protein Recognition.

Protein-protein interaction networks are critical components of cellular regulation. Hub proteins, defined by their ability to interact with numerous protein partners, are the pivots of these networks. A hypothesis that an ensemble of rapidly interconverting conformational states contributes significantly to the ability of hub proteins to interact with diverse partners has been proposed. The master gene regulator p53 is a prototype multidomain hub protein. Its DNA-binding domain alone is involved in interactions with many of its partner proteins. We investigated the dynamics of the p53 DNA-binding domain by 15N-NMR Carr-Purcell-Meiboom-Gill relaxation methods. In the DNA-bound state, we detected conformational exchanges in the domain in the microsecond to millisecond timescale, while dynamics at this timescale was not detectable in the free state. This suggests that the binding of p53 to specific DNA sequences promotes exchange between two or more conformational states, creating a broad conformational repertoire necessary for interacting with many partner proteins.

Dynamics and electrostatics define an allosteric druggable site within the receptor-binding domain of SARS-CoV-2 spike protein.

The pathogenesis of the SARS-CoV-2 virus initiates through recognition of the angiotensin-converting enzyme 2 (ACE2) receptor of the host cells by the receptor-binding domain (RBD) located at the spikes of the virus. Here, using molecular dynamics simulations, we have demonstrated the allosteric crosstalk within the RBD in the apo- and the ACE2 receptor-bound states, revealing the contribution of the dynamics-based correlated motions and the electrostatic energy perturbations to this crosstalk. While allostery, based on correlated motions, dominates inherent distal communication in the apo-RBD, the electrostatic energy perturbations determine favorable pairwise crosstalk within the RBD residues upon binding to ACE2. Interestingly, the allosteric path is composed of residues which are evolutionarily conserved within closely related coronaviruses, pointing toward the biological relevance of the communication and its potential as a target for drug development.

Toward COVID-19 Therapeutics: A Viewpoint from the Nonprotein Amino Acid Based Synthetic Peptide Design Approach.

Cell entry, the fundamental step in cross-species transmission of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), is initiated by the recognition of the host cell angiotensin-converting enzyme-2 (ACE2) receptor by the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. To date, several peptides have been proposed against SARS-CoV-2 both as inhibitor agents or as detection tools that can also be attached to the surfaces of nanoparticle carriers. But owing to their natural amino acid sequences, such peptides cannot be considered as efficient therapeutic candidates from a biostability point of view. This discussion demonstrates the design strategy of synthetic nonprotein amino acid substituted peptides with enhanced biostability and binding affinity, the implication of which can make those peptides potential therapeutic agents for inhibition and simple detection tools.

Heptameric Peptide Interferes with Amyloid-ß Aggregation by Structural Reorganization of the Toxic Oligomers.

Pathogenesis of Alzheimer's disease (AD), the most common type of dementia, involves misfolding and aggregation of the extracellular amyloid-ß (Aß) protein where the intermediate oligomers, formed during the aggregation progression cascade, are considered the prime toxic species. Here, we identify an active peptide fragment from a medicinal plant-derived (Aristolochia indica) fibrinolytic enzyme having anti-amyloidogenic effects against Aß fibrillation and toxicity. Liquid chromatography with tandem mass spectrometry (LC-MS/MS), followed by computational analysis of the peptide pool generated by proteolytic digestion of the enzyme, identifies two peptide sequences with predictive high-propensity binding to Aß42. Microscopic visualizations in conjunction with biochemical and biophysical assessments suggest that the synthetic version of one of the peptides (termed here Pactive, GFLLHQK) arrests Aß molecules in off-pathway oligomers that can no longer participate in the cytotoxic fibrillation pathway. In contrast, the other peptide (termed P1) aggravates the fibrillation process. Further investigations confirm the strong binding affinity of Pactive with both Aß42 monomers and toxic oligomers by biolayer interferometric assays. We have also shown that, mechanistically, Pactive binding induces conformational alterations in the Aß molecule along with modification of Aß hydrophobicity, one of the key players in aggregation. Importantly, the biostability of Pactive in human blood serum and its nontoxic nature make it a promising therapeutic candidate against Alzheimer's, for which no disease-modifying treatments are available to date.

The cancer-associated, gain-of-function TP53 variant P152Lp53 activates multiple signaling pathways implicated in tumorigenesis.

TP53 is the most frequently mutated tumor suppressor gene in many cancers, yet biochemical characterization of several of its reported mutations with probable biological significance have not been accomplished enough. Specifically, missense mutations in TP53 can contribute to tumorigenesis through gain-of-function of biochemical and biological properties that stimulate tumor growth. Here, we identified a relatively rare mutation leading to a proline to leucine substitution (P152L) in TP53 at the very end of its DNA-binding domain (DBD) in a sample from an Indian oral cancer patient. Although the P152Lp53 DBD alone bound to DNA, the full-length protein completely lacked binding ability at its cognate DNA motifs. Interestingly, P152Lp53 could efficiently tetramerize, and the mutation had only a limited impact on the structure and stability of full-length p53. Significantly, when we expressed this variant in a TP53-null cell line, it induced cell motility, proliferation, and invasion compared with a vector-only control. Also, enhanced tumorigenic potential was observed when P152Lp53-expressing cells were xenografted into nude mice. Investigating the effects of P152Lp53 expression on cellular pathways, we found that it is associated with up-regulation of several pathways, including cell-cell and cell-extracellular matrix signaling, epidermal growth factor receptor signaling, and Rho-GTPase signaling, commonly active in tumorigenesis and metastasis. Taken together, our findings provide a detailed account of the biochemical and cellular alterations associated with the cancer-associated P152Lp53 variant and establish it as a gain-of-function TP53 variant.

A Potent Conformation-Constrained Synthetic Peptide Mimic of a Homeodomain Selectively Regulates Target Genes in Cells.

DNA, as a target for therapeutic intervention, remains largely unexplored. DLX-4, a homeodomain containing transcription factor, and its spliced isoforms play crucial roles in many aspects of cellular biochemistry and important roles in many diseases. A smaller peptide mimicking the homeodomain of the transcription factor DLX-4 was designed and synthesized by suitable conjoining of its modified DNA-binding elements. The peptide binds to DLX-4 target sites on the regulatory region of the globin gene cluster with native-like affinity and specificity in vitro. When conjugated to cell penetrating and nuclear localization sequences, it upregulated some of the genes repressed by DLX-4 or its isoforms, such as ß- and γ-globin genes in erythropoietin-induced differentiating CD34+ human hematopoietic stem/progenitor cells with high specificity by competing with the respective binding sites. Engineered peptides mimicking DNA-binding domains of transcription factors offer the potential for creating synthetic molecules for directly targeting DNA sites with high specificity.