Identification of New In Vivo TonB-FepA Rendezvous Sites

The TonB system of Gram-negative bacteria uses the protonmotive force of the cytoplasmic membrane to energize active transport of large or scarce nutrients across the outer membrane by means of customized beta-barrels known as TonB-dependent transporters (TBDTs). The lumen of each TBDT is occluded by an amino-terminal domain, called the cork, which must be displaced for transport of nutrients or translocation of the large protein toxins that parasitize the system. A complex of cytoplasmic membrane proteins consisting of TonB, ExbB and ExbD harnesses the protonmotive force that TonB transmits to the TBDT. The specifics of this energy transformation are a source of continuing interest. The amino terminal domain of a TBDT contains a region called the TonB box, that is essential for the reception of energy from TonB. This domain is the only identified site of in vivo interaction between the TBDT and TonB, occurring through a non-essential region centered on TonB residue Q160. Because TonB binds to TBDTs whether or not it is active or even intact, the mechanism and extent of cork movement in vivo has been challenging to discover. In this study, we used in vivo disulfide crosslinking between eight engineered Cys residues in Escherichia coli TonB and 42 Cys substitutions in the TBDT FepA, including the TonB box, to identify novel sites of interaction in vivo. The TonB Cys substitutions in the core of an essential carboxy terminal amphipathic helix (residues 199-216) were compared to TonB Q160C interactions. Functionality of the in vivo interactions was established when the presence of the inactive TonB H20A mutation inhibited them. A previously unknown functional interaction between the hydrophilic face of the amphipathic helix and the FepA TonB box was identified. Interaction of Q160C with the FepA TonB box appeared to be less functionally important. The two different parts of TonB also differed in their interactions with the FepA cork and barrel turns. While the TonB amphipathic helix Cys residues interacted only with Cys residues on the periplasmic face of the FepA cork, TonB Q160C interacted with buried Cys substitutions within the FepA cork, the first such interactions seen with any TBDT. Both sets of interactions required active TonB. Taken together, these data suggest a model where the amphipathic helix binds to the TonB box, causing the mechanically weak domain of the FepA cork to dip sufficiently into the periplasmic space for interaction with the TonB Q160 region, which is an interaction that does not occur if the TonB box is deleted. The TonB amphipathic helix also interacted with periplasmic turns between FepA β-strands in vivo supporting a surveillance mechanism where TonB searched for TBDTs on the periplasmic face of the outer membrane.

A SARS-CoV-2 mutant from B.1.258 lineage with ∆H69/∆V70 deletion in the Spike protein circulating in Central Europe in the fall 2020

SARS-CoV-2 mutants carrying the ∆H69/∆V70 deletion in the amino-terminal domain of the Spike protein emerged independently in at least six lineages of the virus (namely, B.1.1.7, B.1.1.298, B.1.160, B.1.177, B.1.258, B.1.375). We analyzed SARS-CoV-2 samples collected from various regions of Slovakia between November and December 2020 that were presumed to contain B.1.1.7 variant due to drop-out of the Spike gene target in an RT-qPCR test caused by this deletion. Sequencing of these samples revealed that although in some cases the samples were indeed confirmed as B.1.1.7, a substantial fraction of samples contained another ∆H69/∆V70 carrying mutant belonging to the lineage B.1.258, which has been circulating in Central Europe since August 2020, long before the import of B.1.1.7. Phylogenetic analysis shows that the early sublineage of B.1.258 acquired the N439K substitution in the receptor-binding domain (RBD) of the Spike protein and, later on, also the deletion ∆H69/∆V70 in the Spike N-terminal domain (NTD). This variant was particularly common in several European countries including the Czech Republic and Slovakia but has been quickly replaced by B.1.1.7 early in 2021.

Membrane Interactions Accelerate the Self-Aggregation of Huntingtin Exon 1 Fragments in a Polyglutamine Length-Dependent Manner

The accumulation of aggregated protein is a typical hallmark of many human neurodegenerative disorders, including polyglutamine-related diseases such as chorea Huntington. Misfolding of the amyloidogenic proteins gives rise to self-assembled complexes and fibres. The huntingtin protein is characterised by a segment of consecutive glutamines which, when exceeding ~ 37 residues, results in the occurrence of the disease. Furthermore, it has also been demonstrated that the 17-residue amino-terminal domain of the protein (htt17), located upstream of this polyglutamine tract, strongly correlates with aggregate formation and pathology. Here, we demonstrate that membrane interactions strongly accelerate the oligomerisation and β-amyloid fibril formation of htt17-polyglutamine segments. By using a combination of biophysical approaches, the kinetics of fibre formation is investigated and found to be strongly dependent on the presence of lipids, the length of the polyQ expansion, and the polypeptide-to-lipid ratio. Finally, the implications for therapeutic approaches are discussed.

Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.

Vaccine-induced mutation in SARS-CoV-2, a matter of concern? (Preprint)

UNSTRUCTURED The emergence of new lineages of corona virus over the different continents has illustrated a significant public health concern. These new strains have a higher rate of transmissibility and have become dominant within a short period of time. The virus strains were also witnessed with an extensive range of mutations in the spike (S) protein, particularly in the receptor-binding domain (RBD) and amino-terminal domain (NTD). The studies concerning the genomic epidemiology of the COVID-19 virus have assisted the scientific community to unveil the evolutionary aspect of viruses and track the transmission dynamics over the world. At present all the mutations are vaccine sensitive, but vaccine-resistant variety may emerge at any time. It is very important that we should close the door quickly, although, strategic problems are there. Mutations are more possible in partially vaccinated areas rather than fully vaccinated areas. The present policy of vaccinating on an age structure leaves many non-vaccinated in the same home. In a population with very few vaccinated people there are viruses in many hosts and the chances of mutation is high. If the population is fully vaccinated, the virus is eradicated from that community and hence mutation chances are less. This locality specific fast and total vaccination will curtail the mutagenesity of the virus, lead to its eradication and to be preferred rather than vaccinating based on age structure.

Conformational rearrangement of the NMDA receptor amino-terminal domain during activation and allosteric modulation

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for synaptic plasticity and memory. Receptor activation involves glycine- and glutamate-stabilized closure of the GluN1 and GluN2 subunit ligand binding domains that is allosterically regulated by the amino-terminal domain (ATD). Using single molecule fluorescence resonance energy transfer (smFRET) to monitor subunit rearrangements in real-time, we observe a stable ATD inter-dimer distance in the Apo state and test the effects of agonists and antagonists. We find that GluN1 and GluN2 have distinct gating functions. Glutamate binding to GluN2 subunits elicits two identical, sequential steps of ATD dimer separation. Glycine binding to GluN1 has no detectable effect, but unlocks the receptor for activation so that glycine and glutamate together drive an altered activation trajectory that is consistent with ATD dimer separation and rotation. We find that protons exert allosteric inhibition by suppressing the glutamate-driven ATD separation steps, and that greater ATD separation translates into greater rotation and higher open probability.