Epidemiological Features of Human Norovirus Genotypes before and after COVID-19 Countermeasures in Osaka, Japan.

We investigated the molecular epidemiology of human norovirus (HuNoV) in all age groups using samples from April 2019 to March 2023, before and after the COVID-19 countermeasures were implemented. GII.2[P16] and GII.4[P31], the prevalent strains in Japan before COVID-19 countermeasures, remained prevalent during the COVID-19 pandemic, except from April to November 2020; in 2021, the prevalence of GII.2[P16] increased among children. Furthermore, there was an increase in the prevalence of GII.4[P16] after December 2022. Phylogenetic analysis of GII.P31 RdRp showed that some strains detected in 2022 belonged to a different cluster of other strains obtained during the present study period, suggesting that HuNoV strains will evolve differently even if they have the same type of RdRp. An analysis of the amino acid sequence of VP1 showed that some antigenic sites of GII.4[P16] were different from those of GII.4[P31]. The present study showed high infectivity of HuNoV despite the COVID-19 countermeasures and revealed changes in the prevalent genotypes and mutations of each genotype. In the future, we will investigate whether GII.4[P16] becomes more prevalent, providing new insights by comparing the new data with those analyzed in the present study.

Crystal structure of the lipid flippase MurJ in a "squeezed" form distinct from its inward- and outward-facing forms.

The bacterial peptidoglycan enclosing the cytoplasmic membrane is a fundamental cellular architecture. The integral membrane protein MurJ plays an essential role in flipping the cell wall building block Lipid II across the cytoplasmic membrane for peptidoglycan biosynthesis. Previously reported crystal structures of MurJ have elucidated its V-shaped inward- or outward-facing forms with an internal cavity for substrate binding. MurJ transports Lipid II using its cavity through conformational transitions between these two forms. Here, we report two crystal structures of inward-facing forms from Arsenophonus endosymbiont MurJ and an unprecedented crystal structure of Escherichia coli MurJ in a "squeezed" form, which lacks a cavity to accommodate the substrate, mainly because of the increased proximity of transmembrane helices 2 and 8. Subsequent molecular dynamics simulations supported the hypothesis that the squeezed form is an intermediate conformation. This study fills a gap in our understanding of the Lipid II flipping mechanism.

Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake.

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

Structural Basis of Sarco/Endoplasmic Reticulum Ca2+-ATPase 2b Regulation via Transmembrane Helix Interplay.

Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and the N-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions.

Remote Coupled Drastic ß-Barrel to ß-Sheet Transition of the Protein Translocation Motor.

The membrane protein SecDF, belonging to the RND superfamily, enhances protein translocation at the extracytoplasmic side using a proton gradient. Here, we report the crystal structure of SecDF in a form we named Super-membrane-facing (Super F) form, demonstrating a ß-barrel architecture instead of the previously reported ß-sheet structure. Through this structural insight and supporting results of an in vivo crosslinking experiment, we propose a remote coupling model in which a structural change of the transmembrane region drives a functional, extracytoplasmic conformational transition.

Tunnel Formation Inferred from the I-Form Structures of the Proton-Driven Protein Secretion Motor SecDF.

Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF.