Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane beta barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TspB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic beta hairpin immediately preceding the first barrel strand can insert into the beta barrel. We propose that these motions enlarge the channel and may hoist the substrate into it for secretion. An anchor region at the interface of the beta barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these proteins mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

Mutation Sites Are Distant from Remdesivir Binding Site in Human SARS-CoV-2 RNA-Dependent RNA Polymerase

<p>The comparison of 10,929 human SARS-CoV-2 RdRp protein sequences representing six geographical locations with the reference protein sequence in human SARS-CoV-2 genome isolate from Wuhan, China, identified 222 distinct mutation sites in the RdRp protein. The NiRAN and interface domains, Fingers, Palm and Thumb sub-domains were each associated with ~20% or more mutations compared to mutations in N-terminal, beta-hairpin or C-terminal regions of the protein. The Pro4715Leu mutation was predominantly observed in RdRp proteins from all six geographical locations; Africa, Asia, Europe, North America, Oceania and South America. None of the mutation site residues were within 3.2 Å interacting distance from remdesivir as observed in the three-dimensional cryo-electron microscopy structures of RdRp protein complexes available in the Protein Data Bank. Therefore, the mutations in human SARS-CoV-2 RdRp proteins, described in the present work, are not likely to cause resistance to remdesivir binding. Further, the mutations were also not associated with functionally important residues that would affect the enzyme’s function.</p>

Mutation Sites Are Distant from Remdesivir Binding Site in Human SARS-CoV-2 RNA-Dependent RNA Polymerase

<p>The comparison of 10,929 human SARS-CoV-2 RdRp protein sequences representing six geographical locations with the reference protein sequence in human SARS-CoV-2 genome isolate from Wuhan, China, identified 222 distinct mutation sites in the RdRp protein. The NiRAN and interface domains, Fingers, Palm and Thumb sub-domains were each associated with ~20% or more mutations compared to mutations in N-terminal, beta-hairpin or C-terminal regions of the protein. The Pro4715Leu mutation was predominantly observed in RdRp proteins from all six geographical locations; Africa, Asia, Europe, North America, Oceania and South America. None of the mutation site residues were within 3.2 Å interacting distance from remdesivir as observed in the three-dimensional cryo-electron microscopy structures of RdRp protein complexes available in the Protein Data Bank. Therefore, the mutations in human SARS-CoV-2 RdRp proteins, described in the present work, are not likely to cause resistance to remdesivir binding. Further, the mutations were also not associated with functionally important residues that would affect the enzyme’s function.</p>

A systematic analysis of the beta hairpin motif in the Protein Data Bank

The beta hairpin motif is a ubiquitous protein structural motif that can be found in molecules across the tree of life. This motif, which is also popular in synthetically designed proteins and peptides, is known for its stability and adaptability to broad functions. Here we systematically probe all 49,000 unique beta hairpin substructures contained within the Protein Data Bank (PDB) to uncover key characteristics correlated with stable beta hairpin structure, including amino acid biases and enriched inter-strand contacts. We also establish a set of broad design principles that can be applied to the generation of libraries encoding proteins or peptides containing beta hairpin structures.ImportanceThe beta hairpin motif is a common protein structural motif that is known for its stability and varied activity in diverse proteins. Here we use nearly fifty thousand beta hairpin substructures from the Protein Data Bank to systematically analyze and identify key characteristics of the beta hairpin motif. Ultimately, we provide a set of design principles for the generation of synthetic libraries encoding proteins containing beta hairpin structures.

Convergence of a common solution to broad ebolavirus neutralization by glycan cap directed human antibodies

SummaryAntibodies that target the glycan cap epitope on ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization is not well-understood. Here we present cryo-electron microscopy (cryo-EM) structures of several glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLD) to the glycan cap, which we name the MLD-anchor and cradle. Antibodies that bind to the MLD-cradle share common features, including the use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form beta-hairpin structures to mimic the MLD-anchor, disrupt MLD attachment, destabilize GP quaternary structure and block cleavage events required for receptor binding. Our results collectively provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies.

Exploring the F-actin/CPEB3 interaction and its possible role in the molecular mechanism of long-term memory

Dendritic spines are tiny membranous protrusions on the dendrites of neurons. Dendritic spines change shape in response to input signals, thereby strengthening the connections between neurons. The growth and stabilization of dendritic spines is thought to be essential for maintaining long-term memory. Actin cytoskeleton remodeling in spines is a key element of their formation and growth. More speculatively, the aggregation of CPEB3, a functional prion that binds RNA, has been reported to be involved in the maintenance of long-term memory. Here we study the interaction between actin and CPEB3 and propose a molecular model for the complex structure of CPEB3 and an actin filament (F-actin). The results of our computational modeling, including both energetic and structural analyses, are compared with novel data from peptide array experiments. Our model of the CPEB3/F-actin interaction suggests that F-actin potentially triggers the aggregation-prone structural transition of a short CPEB3 sequence by zipping it into a beta-hairpin form. We also propose that the CPEB3/F-actin interaction might be regulated by the SUMOylation of CPEB3, based on bioinformatic searches for potential SUMOylation sites as well as SUMO interacting motifs in CPEB3. On the basis of these results and the existing literature, we put forward a possible molecular mechanism underlying long-term memory that involves CPEB3’s binding to actin, its aggregation, and its regulation by SUMOylation.

Expression of Thanatin in HEK293 Cells and Investigation of its Antibacterial Effects on Some Human Pathogens

Background: Thanatin is the smallest member of Beta-hairpin class of cationic peptide derived from insects with vast activities against various pathogens. Objective: n this study, the antimicrobial activity of this peptide against some species of human bacterial pathogens as well as its toxicity on NIH cells were evaluated. Method: Thanatin DNA sequence was cloned into pcDNA3.1+ vector and transformed into a DH5α bacterial strain. Then the recombinant plasmids were transfected into HEK-293 cells by calcium phosphate co-precipitation. After applying antibiotic treatment, the supernatant medium containing thanatin was collected. The peptide quantity was estimated by SDS-PAGE and GelQuant software. The antimicrobial activity of this peptide was performed with Minimum Inhibitory Concentration (MIC) method. In addition, its toxicity on NIH cells were evaluated by MTT assay. Results: The peptide quantity was estimated approximately 164.21 µmolL-1. The antibacterial activity of thanatin was estimated between 0.99 and 31.58 µmolL-1 using MIC method. The result of cytotoxicity test on NIH cell line showed that the peptide toxicity up to the concentration of 394.10 µmolL-1 and for 48 hours, was not statistically significant from negative control cells (P>0.05). The antimicrobial assay demonstrated that thanatin had an antibacterial effect on some tested microorganisms. The results obtained in this study also showed that thanatin had no toxicity on mammalian cell lines including HEK293 and NIH. Conclusion: Antimicrobial peptides such as thanatin are considered to be appropriate alternatives to conventional antibiotics in treating various human pathological diseases bacteria.

Origin of the Genetic Code Is Found at the Transition between a Thioester World of Peptides and the Phosphoester World of Polynucleotides

The early metabolism arising in a Thioester world gave rise to amino acids and their simple peptides. The catalytic activity of these early simple peptides became instrumental in the transition from Thioester World to a Phosphate World. This transition involved the appearances of sugar phosphates, nucleotides, and polynucleotides. The coupling of the amino acids and peptides to nucleotides and polynucleotides is the origin for the genetic code. Many of the key steps in this transition are seen in in the catalytic cores of the nucleotidyltransferases, the class II tRNA synthetases (aaRSs) and the CCA adding enzyme. These catalytic cores are dominated by simple beta hairpin structures formed in the Thioester World. The code evolved from a proto-tRNA a tetramer XCCA interacting with a proto-aminoacyl-tRNA synthetase (aaRS) activating Glycine and Proline, the initial expanded code is found in the acceptor arm of the tRNA, the operational code. It is the coevolution of the tRNA with the aaRSs that is at the heart of the origin and evolution of the genetic code. There is also a close relationship between the accretion models of the evolving tRNA and that of the ribosome.