Structural Analyses of Designed α-Helix and ß-Sheet Peptide Nanofibers Using Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy and Introduction of Structure-Based Metal-Responsive Properties.

The 21-residue peptide α3, which is artificially designed and consists of three repeats of 7 residues, is known to rapidly assemble into the α-helix nanofiber. However, its molecular structure within the fiber has not yet been fully elucidated. Thus, we conducted a thorough investigation of the fiber's molecular structure using solid-state NMR and other techniques. The molecules were found to be primarily composed of the α-helix structure, with some regions near the C- and N-terminal adopting a 310-helix structure. Furthermore, it was discovered that ß-sheet hydrogen bonds were formed between the molecules at both ends. These intermolecular interactions caused the molecules to assemble parallelly in the same direction, forming helical fibers. In contrast, we designed two molecules, CaRP2 and ßKE, that can form ß-sheet intermolecular hydrogen bonds using the entire molecule instead of just the ends. Cryo-EM and other measurements confirmed that the nanofibers formed in a cross ß structure, albeit at a slow rate, with the formation times ranging from 1 to 42 days. To create peptide nanofibers that instantaneously respond to changes in the external environment, we designed several molecules (HDM1-3) based on α3 by introducing metal-binding sites. One of these molecules was found to be highly responsive to the addition of metal ions, inducing α-helix formation and simultaneously assembling into nanofibers. The nanofibers lost their structure upon removal of the metal ion. The change occurred promptly and was reversible, demonstrating that the intended level of responsiveness was attained.

Combined computed tomography and C-arm resuscitation room system (CTCARM) is associated with decreased time to definitive hemostasis and reduces preperitoneal pelvic packing maneuvers in severe pelvic trauma.

Objectives: Severe pelvic fracture concomitant with massive bleeding is potentially lethal, and intervention for hemorrhage control still depends on institutional supplies. With the recent installation of a CT and C-arm combined resuscitation room system (CTCARM) for treatment of trauma patients in our institution, the strategic process and options for hemorrhage control after pelvic fracture have changed. We retrospectively reviewed the procedures we performed and their outcomes. Methods: The CTCARM was installed in our trauma resuscitation room in April 2020. Patients who were diagnosed as having pelvic fracture and underwent interventional radiology for hemorrhage control within 2.5 hours after arrival were compared before and after CTCARM installation. We reviewed the time process for hemorrhage control, treatment options performed, blood products used and their outcomes. Results: Included in this study were 56 patients treated between 2016 and 2022, of whom 36 patients were treated before (original group) and 20 patients after CTCARM installation (CTCARM group). Patient characteristics and vital signs at admission were not statistically different. Preperitoneal pelvic packing was performed significantly more frequently in the original group (p<0.01), whereas resuscitative endovascular balloon occlusion of the aorta use was much more frequent in the CTCARM group (p=0.02). Although the times from admission to first angiography (p=0.014) and to complete hemostasis (p=0.02) were significantly shorter in the CTCARM group, mortality was not statistically different. Four preventable trauma deaths occurred in the original group, but there were none in the CTCARM group. Six unexpected survivors were observed in the original group and four in the CTCARM group. Conclusions: Although the CTCARM had no direct effects on patient mortality for now, it has allowed us to accelerate the treatment time process, shorten preperitoneal pelvic packing procedural time, and potentially avoid subsequent preventable trauma deaths. Level of evidence: Level IV.

Factors associated with the outcome of patients with COVID-19 requiring mechanical ventilation: A single-center observational study in Japan.

Aim: Coronavirus disease (COVID-19) spread worldwide, and was declared as a pandemic by the World Health Organization. Despite numerous studies in the last few years, the factors associated with the outcomes of patients with COVID-19 requiring mechanical ventilation remain unclear. The prediction of ventilator weaning and mortality using the data obtained at the time of intubation could be beneficial for establishing appropriate treatment strategies and obtaining informed consent. In this study, we aimed to clarify the association between patient information at the time of intubation and the outcomes of intubated COVID-19 patients. Methods: This retrospective observational study used single-center data from patients with COVID-19. Patients with COVID-19 who were admitted to Osaka Metropolitan University Hospital from April 1, 2020, to March 31, 2022, and under mechanical ventilation were included. The main outcome was defined as the factors related to ventilator weaning; a multivariate analysis was carried out to evaluate the association between patient information at the time of intubation and the outcome. Results: In total, 146 patients were included in this study. The factors significantly associated with ventilator weaning were age (65-74 years old, adjusted odds ratio [OR], 0.168; 75 years and older, adjusted OR, 0.121), vaccination history (adjusted OR, 5.655), and Sequential Organ Failure Assessment (SOFA) respiration score (adjusted OR, 0.007) at the time of intubation. Conclusion: Age, SOFA respiration score, and COVID-19 vaccination history at the time of intubation could be associated with outcomes in patients with COVID-19 requiring mechanical ventilation.

Three structures of PSI-LHCI from Chlamydomonas reinhardtii suggest a resting state re-activated by ferredoxin.

Photosystem I (PSI) from the green alga Chlamydomonas reinhardtii, with various numbers of membrane bound antenna complexes (LHCI), has been described in great detail. In contrast, structural characterization of soluble binding partners is less advanced. Here, we used X-ray crystallography and single particle cryo-EM to investigate three structures of the PSI-LHCI supercomplex from Chlamydomonas reinhardtii. An X-ray structure demonstrates the absence of six chlorophylls from the luminal side of the LHCI belts, suggesting these pigments were either physically absent or less stably associated with the complex, potentially influencing excitation transfer significantly. CryoEM revealed extra densities on luminal and stromal sides of the supercomplex, situated in the vicinity of the electron transfer sites. These densities disappeared after the binding of oxidized ferredoxin to PSI-LHCI. Based on these structures, we propose the existence of a PSI-LHCI resting state with a reduced active chlorophyll content, electron donors docked in waiting positions and regulatory binding partners positioned at the electron acceptor site. The resting state PSI-LHCI supercomplex would be recruited to its active form by the availability of oxidized ferredoxin.

Filamentous structures in the cell envelope are associated with bacteroidetes gliding machinery.

Many bacteria belonging to the phylum Bacteroidetes move on solid surfaces, called gliding motility. In our previous study with the Bacteroidetes gliding bacterium Flavobacterium johnsoniae, we proposed a helical loop track model, where adhesive SprB filaments are propelled along a helical loop on the cell surface. In this study, we observed the gliding cell rotating counterclockwise about its axis when viewed from the rear to the advancing direction of the cell and revealed that one labeled SprB focus sometimes overtook and passed another SprB focus that was moving in the same direction. Several electron microscopic analyses revealed the presence of a possible multi-rail structure underneath the outer membrane, which was associated with SprB filaments and contained GldJ protein. These results provide insights into the mechanism of Bacteroidetes gliding motility, in which the SprB filaments are propelled along tracks that may form a multi-rail system underneath the outer membrane. The insights may give clues as to how the SprB filaments get their driving force.

Isolation and structure of the fibril protein, a major component of the internal ribbon for Spiroplasma swimming.

Spiroplasma, which are known pathogens and commensals of arthropods and plants, are helical-shaped bacteria that lack a peptidoglycan layer. Spiroplasma swim by alternating between left- and right-handed helicity. Of note, this system is not related to flagellar motility, which is widespread in bacteria. A helical ribbon running along the inner side of the helical cell should be responsible for cell helicity and comprises the bacterial actin homolog, MreB, and a protein specific to Spiroplasma, fibril. Here, we isolated the ribbon and its major component, fibril filament, for electron microscopy (EM) analysis. Single-particle analysis of the fibril filaments using the negative-staining EM revealed a three-dimensional chain structure composed of rings with a size of 11 nm wide and 6 nm long, connected by a backbone cylinder with an 8.7 nm interval with a twist along the filament axis. This structure was verified through EM tomography of quick-freeze deep-etch replica sample, with a focus on its handedness. The handedness and pitch of the helix for the isolated ribbon and fibril filament agreed with those of the cell in the resting state. Structures corresponding to the alternative state were not identified. These results suggest that the helical cell structure is supported by fibril filaments; however, the helical switch is caused by the force generated by the MreB proteins. The isolation and structural outline of the fibril filaments provide crucial information for an in-depth clarification of the unique swimming mechanism of Spiroplasma.

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile.

Some bacteria express a binary toxin translocation system, consisting of an enzymatic subunit and translocation pore, that delivers enzymes into host cells through endocytosis. The most clinically important bacterium with such a system is Clostridioides difficile (formerly Clostridium). The CDTa and CDTb proteins from its system represent important therapeutic targets. CDTb has been proposed to be a di-heptamer, but its physiological heptameric structure has not yet been reported. Here, we report the cryo-EM structure of CDTa bound to the CDTb-pore, which reveals that CDTa binding induces partial unfolding and tilting of the first CDTa α-helix. In the CDTb-pore, an NSS-loop exists in 'in' and 'out' conformations, suggesting its involvement in substrate translocation. Finally, 3D variability analysis revealed CDTa movements from a folded to an unfolded state. These dynamic structural information provide insights into drug design against hypervirulent C. difficile strains.

Structure of cyanobacterial photosystem I complexed with ferredoxin at 1.97 Å resolution.

Photosystem I (PSI) is a light driven electron pump transferring electrons from Cytochrome c6 (Cyt c6) to Ferredoxin (Fd). An understanding of this electron transfer process is hampered by a paucity of structural detail concerning PSI:Fd interface and the possible binding sites of Cyt c6. Here we describe the high resolution cryo-EM structure of Thermosynechococcus elongatus BP-1 PSI in complex with Fd and a loosely bound Cyt c6. Side chain interactions at the PSI:Fd interface including bridging water molecules are visualized in detail. The structure explains the properties of mutants of PsaE and PsaC that affect kinetics of Fd binding and suggests a molecular switch for the dissociation of Fd upon reduction. Calorimetry-based thermodynamic analyses confirms a single binding site for Fd and demonstrates that PSI:Fd complexation is purely driven by entropy. A possible reaction cycle for the efficient transfer of electrons from Cyt c6 to Fd via PSI is proposed.

Structures of multisubunit membrane complexes with the CRYO ARM 200.

Progress in structural membrane biology has been significantly accelerated by the ongoing 'Resolution Revolution' in cryo-electron microscopy (cryo-EM). In particular, structure determination by single-particle analysis has evolved into the most powerful method for atomic model building of multisubunit membrane protein complexes. This has created an ever-increasing demand in cryo-EM machine time, which to satisfy is in need of new and affordable cryo-electron microscopes. Here, we review our experience in using the JEOL CRYO ARM 200 prototype for the structure determination by single-particle analysis of three different multisubunit membrane complexes: the Thermus thermophilus V-type ATPase VO complex, the Thermosynechococcus elongatus photosystem I monomer and the flagellar motor lipopolysaccharide peptidoglycan ring (LP ring) from Salmonella enterica.

Structural insight into the activation mechanism of MrgD with heterotrimeric Gi-protein revealed by cryo-EM.

MrgD, a member of the Mas-related G protein-coupled receptor (MRGPR) family, has high basal activity for Gi activation. It recognizes endogenous ligands, such as ß-alanine, and is involved in pain and itch signaling. The lack of a high-resolution structure for MrgD hinders our understanding of whether its activation is ligand-dependent or constitutive. Here, we report two cryo-EM structures of the MrgD-Gi complex in the ß-alanine-bound and apo states at 3.1 Å and 2.8 Å resolution, respectively. These structures show that ß-alanine is bound to a shallow pocket at the extracellular domains. The extracellular half of the sixth transmembrane helix undergoes a significant movement and is tightly packed into the third transmembrane helix through hydrophobic residues, creating the active form. Our structures demonstrate a structural basis for the characteristic ligand recognition of MrgD. These findings provide a framework to guide drug designs targeting the MrgD receptor.

Structural insights into blue-green light utilization by marine green algal light harvesting complex II at 2.78 Å.

Light-harvesting complex II (LHCII) present in plants and green algae absorbs solar energy to promote photochemical reactions. A marine green macroalga, Codium fragile, exhibits the unique characteristic of absorbing blue-green light from the sun during photochemical reactions while being underwater owing to the presence of pigment-altered LHCII called siphonaxanthin-chlorophyll a/b-binding protein (SCP). In this study, we determined the structure of SCP at a resolution of 2.78 Å using cryogenic electron microscopy. SCP has a trimeric structure, wherein each monomer containing two lutein and two chlorophyll a molecules in the plant-type LHCII are replaced by siphonaxanthin and its ester and two chlorophyll b molecules, respectively. Siphonaxanthin occupies the binding site in SCP having a polarity in the trimeric inner core, and exhibits a distorted conjugated chain comprising a carbonyl group hydrogen bonded to a cysteine residue of apoprotein. These features suggest that the siphonaxanthin molecule is responsible for the characteristic green absorption of SCP. The replaced chlorophyll b molecules extend the region of the stromal side chlorophyll b cluster, spanning two adjacent monomers.

Chained Structure of Dimeric F1-like ATPase in Mycoplasma mobile Gliding Machinery.

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called "chains" that are each composed of 17 repeating protein units called "particles." These proteins include homologs of the catalytic α and ß subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (ß-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αß)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αß)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCE F1Fo-ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

Native flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries.

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.

Total Synthesis and Structural Characterization of Caveolin-1.

Caveolin-1, which is an essential protein for caveola formation, was chemically synthesized. It is composed of 177 amino acid residues, is triply palmitoylated at the C-terminal region, and is inserted into the lipid bilayer to form a V-shaped structure in the middle of the polypeptide chain. The entire sequence was divided into five peptide segments, each of which was synthesized by the solid-phase method. To improve the solubility of the C-terminal region, O-acyl isopeptide structures were incorporated. After ligation by the thioester method and the introduction of the palmitoyl groups, all the protecting groups were removed and the isopeptide structures were converted into the native peptide bond. Finally, the obtained polypeptide was successfully inserted into bicelles, thus showing the success of the synthesis.

Split conformation of Chaetomium thermophilum Hsp104 disaggregase.

Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 65 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.

Cryo-EM structure of a functional monomeric Photosystem I from Thermosynechococcus elongatus reveals red chlorophyll cluster.

A high-resolution structure of trimeric cyanobacterial Photosystem I (PSI) from Thermosynechococcus elongatus was reported as the first atomic model of PSI almost 20 years ago. However, the monomeric PSI structure has not yet been reported despite long-standing interest in its structure and extensive spectroscopic characterization of the loss of red chlorophylls upon monomerization. Here, we describe the structure of monomeric PSI from Thermosynechococcus elongatus BP-1. Comparison with the trimer structure gave detailed insights into monomerization-induced changes in both the central trimerization domain and the peripheral regions of the complex. Monomerization-induced loss of red chlorophylls is assigned to a cluster of chlorophylls adjacent to PsaX. Based on our findings, we propose a role of PsaX in the stabilization of red chlorophylls and that lipids of the surrounding membrane present a major source of thermal energy for uphill excitation energy transfer from red chlorophylls to P700.

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring.

The bacterial flagellum is a protein nanomachine essential for bacterial motility. The flagellar basal body contains several ring structures. The MS-ring is embedded in the cytoplasmic membrane and is formed at the earliest stage of flagellar formation to serve as the base for flagellar assembly as well as a housing for the flagellar protein export gate complex. The MS-ring is formed by FliF, which has two transmembrane helices and a large periplasmic region. A recent electron cryomicroscopy (cryoEM) study of the MS-ring formed by overexpressed FliF revealed a symmetry mismatch between the S-ring and inner part of the M-ring. However, the actual symmetry relation in the native MS-ring and positions of missing domains remain obscure. Here, we show the structure of the M-ring by combining cryoEM and X-ray crystallography. The crystal structure of the N-terminal half of the periplasmic region of FliF showed that it consists of two domains (D1 and D2) resembling PrgK D1/PrgH D2 and PrgK D2/PrgH D3 of the injectisome. CryoEM analysis revealed that the inner part of the M-ring shows a gear wheel-like density with the inner ring of C23 symmetry surrounded by cogs with C11 symmetry, to which 34 copies of FliFD1-D2 fitted well. We propose that FliFD1-D2 adopts two distinct orientations in the M-ring relative to the rest of FliF, with 23 chains forming the wheel and 11 chains forming the cogs, and the 34 chains come together to form the S-ring with C34 symmetry for multiple functions of the MS-ring.IMPORTANCE The bacterial flagellum is a motility organelle formed by tens of thousands of protein molecules. At the earliest stage of flagellar assembly, a transmembrane protein, FliF, forms the MS-ring in the cytoplasmic membrane as the base for flagellar assembly. Here, we solved the crystal structure of a FliF fragment. Electron cryomicroscopy (cryoEM) structural analysis of the MS-ring showed that the M-ring and S-ring have different rotational symmetries. By docking the crystal structure of the FliF fragment into the cryoEM density map of the entire MS-ring, we built a model of the whole periplasmic region of FliF and proposed that FliF adopts two distinct conformations to generate three distinct C11, C23, and C34 symmetries within the MS-ring for its multiple functions.

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s.

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

In Situ Structural Analysis of Leptospira spp. by Electron Cryotomography.

Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is capable of determining protein structures at resolutions high enough to visualize their individual domains and secondary structures in their cellular environment.