Biology subjects

Sugita, Y.

Publications and source records attributed to Sugita, Y..

35 records · Page 2Linked to original sources

bioRxiv2024

Structural and Dynamic Insights into the Biased Signaling Mechanism of the Human Kappa Opioid Receptor

The {kappa}-opioid receptor (KOR) is a member of the G protein-coupled receptor (GPCR) family, responsible for modulating cellular responses through transducers such as G proteins and arrestins. G protein-biased KOR agonists hold promise due to their potential to mitigate side effects such as drug aversion and sedation while preserving analgesic and antipruritic effects. Here, we shed light on the structural dynamics of the human KOR-Gi signaling complex bound with either nalfurafine (a G-protein-biased agonist) or U-50,488H (a balanced agonist) using cryo-electron microscopy (cryo-EM). Cryo-EM structures of the KOR-Gi signaling complexes identify the ligand binding mode in the activated state. Vibrational spectroscopy analysis reveals changes in the ligand-binding pocket upon binding to these ligands. Cell-based mutant experiments pinpoint four amino acids (K2275.40, C2866.47, H2916.52, and Y3127.34; Ballesteros-Weinstein numbering is shown in superscript) that play crucial roles in arrestin recruitment. Among these four amino acids, H2916.52 and Y3127.34 are also implicated in G-protein coupling. Our findings pave the way for targeting specific residues in the KOR ligand-binding pocket to enhance KOR-mediated therapeutic effects while mitigating unwanted side effects.

biophysics↗

bioRxiv2024

Structural basis of Ebola virus nucleocapsid assembly and functions

The Ebola virus, a member of the Filoviridae family, causes severe hemorrhagic fever in humans. Filamentous virions contain a helical nucleocapsid responsible for genome transcription, replication, and packaging into progeny virions. The nucleocapsid consists of a helical nucleoprotein (NP)-viral genomic RNA complex forming the core structure, to which VP24 and VP35 bind externally. Two NPs, each paired with a VP24 molecule, constitute a repeating unit. However, the detailed nucleocapsid structure remains unclear. Here, we determined the nucleocapsid-like structure within virus-like particles at 4.6 [A] resolution using single-particle cryo-electron microscopy. Mutational analysis identified specific interactions between the two NPs and two VP24s and demonstrated that each of the two VP24s in different orientations distinctively regulates nucleocapsid assembly, viral RNA synthesis, and intracellular transport of the nucleocapsid. Our findings highlight the sophisticated mechanisms underlying the assembly and functional regulation of the nucleocapsid and provide insights into antiviral development.

microbiology↗

bioRxiv2023

GENESIS CGDYN: large-scale coarse-grained MD simulation with dynamicload balancing for heterogeneous biomolecular systems

Residue-level coarse-grained (CG) molecular dynamics (MD) simulation is widely used to investigate slow biological processes that involve multiple proteins, nucleic acids, and their complexes. Biomolecules in a large simulation system are distributed non-uniformly, limiting computational efficiency with conventional methods. Here, we develop a hierarchical domain decomposition scheme with dynamic load balancing for heterogeneous biomolecular systems to keep computational efficiency even after drastic changes in particle distribution. The new schemes are applied to intrinsically disordered protein (IDP) droplet fusions. The droplet shape changes correlate with mixing IDP chains from two droplets. We also simulate formations of large IDP droplets, whose sizes are almost equivalent to those observed in microscopy. The methods have been implemented in CGDYN of the GENESIS software, which provides a new tool for investigating mesoscopic biological phenomena using the residue-level CG models.

biophysics↗

bioRxiv2023

Micelle-like clusters in phase-separated Nanog condensates: A molecular simulation study

The phase separation model for transcription suggests that transcription factors (TFs), coactivators, and RNA polymerases form biomolecular condensates around active gene loci and regulate transcription. However, the structural details of condensates remain elusive. In this study, for Nanog, a master TF in mammalian embryonic stem cells known to form protein condensates in vitro, we examined protein structures in the condensates using residue-level coarse-grained molecular simulations. Human Nanog formed micelle-like clusters in the condensate. In the micelle-like cluster, the C-terminal disordered domains, including the tryptophan repeat (WR) regions, interacted with each other near the cluster center primarily via hydrophobic interaction. In contrast, hydrophilic disordered N-terminal and DNA-binding domains were exposed on the surface of the clusters. Electrostatic attractions of these surface residues were responsible for bridging multiple micelle-like structures in the condensate. The micelle-like structure and condensate were dynamic and liquid-like. Mutation of tryptophan residues in the WR region which was implicated to be important for a Nanog function resulted in dissolution of the Nanog condensate. Finally, to examine the impact of Nanog cluster to DNA, we added DNA fragments to the Nanog condensate. Nanog DNA-binding domains exposed to the surface of the micelle-like cluster could recruit more than one DNA fragments, making DNA-DNA distance shorter. Author summaryIn eukaryotic transcription regulation, enhancer elements far from the promoter are known to modulate transcriptional activity, but the molecular mechanism remains elusive. One of these models, the phase separation model, suggests that transcription factors (TFs), coactivators, and transcription machinery form biomolecular condensates that include enhancer and promoter elements, making enhancer-promoter communication possible via the protein network in the condensate. Nanog, one of the core TFs involved in mammalian embryonic stem cells, has been reported to have the ability to form condensates. In this study, we addressed the structural details of Nanog condensates by performing residue-level coarse-grained molecular simulations. We found that Nanog formed micelle-like clusters via primarily hydrophobic interactions between tryptophan repeat regions in the C-terminal disordered domains within the condensate. On the other hand, highly charged N-terminal and DNA-binding domains were exposed to the surface of the micelle and were responsible for bridging many micelles into a condensate. The micelle-like clusters and condensate were dynamic and liquid-like. In addition, Nanog condensates could induce DNA-DNA attraction mediated by micelle-like structures.

biophysics↗

bioRxiv2022

Repulsive interaction and secondary structure of highly charged proteins in regulating biomolecular condensation

Biomolecular condensation is involved in various cellular processes both functional and dysfunctional. Regulation of the condensation is thus crucial to avoid pathological protein aggregation and to maintain stable cellular environments. Recently, a class of highly charged intrinsically disordered proteins (IDPs), which are called the heat-resistant obscure (Hero) proteins, are shown to protect other client proteins from pathological aggregation. Besides the potential importance of this function, molecular mechanisms for how Hero proteins can protect other proteins from aggregation are not still known. Here we perform multiscale molecular dynamics (MD) simulations of Hero11, one of the Hero proteins, and the C-terminal region of TDP-43, as a target protein of Hero11, at various conditions to examine how they interact with each other. Based on the simulation results, three possible mechanisms have been proposed: (i) TDP-43 and Hero11 in dense phase reduces contacts with each other and shows faster diffusion due to the repulsive Hero11-Hero11 interactions, (ii) the amount of TDP-43 in dilute phase increases and their sizes become greater upon the attractive Hero11-TDP-43 interactions, and (iii) Hero-11 on the surface of small TDP-43 condensates avoids their fusions with the repulsive interactions. We also examine possible Hero-11 structures in atomistic and coarse-grained MD simulations and found disordered Hero-11 tend to assemble on the surface of the condensates, avoiding the droplet fusion effectively. The proposed mechanisms give us new insight into the regulation of biomolecular condensation in the cells and other conditions.

biophysics↗

bioRxiv2022

The impact of inhibitor size and flexibility on the binding pathways to c-Src kinase

Considering dynamical aspects of protein-drug binding processes is inevitable in current drug compound design. Conformational plasticity of protein kinases poses a challenge for the design of their inhibitors, and therefore, atomistic molecular dynamics (MD) simulations have often been utilized. While protein conformational changes have been increasingly discussed, a fundamental yet non-trivial question remains for the effect of drug compound flexibility, which is hardly detectable from experiments. In this study, we apply two-dimensional replica-exchange MD simulations as enhanced sampling to investigate how c-Src kinase can bind PP1, a small inhibitor, and dasatinib, a larger inhibitor with greater flexibility. 600 microseconds simulations in total sample binding and unbinding events of these inhibitors much more frequently than conventional MD simulation, resulting in statistically converged binding pathways. While the two inhibitors adopt a similar mechanism of multiple binding pathways, the non-canonical binding poses become less feasible for dasatinib. A notable difference is apparent in their energetics where dasatinib stabilizes at intermediate states more than PP1 to raise the barrier toward the canonical pose. Conformational analysis shows that dasatinib adopts linear and bent forms for which relative populations are altered upon binding. We further find hidden conformations of dasatinib at intermediate regions, and unexpectedly one of them could efficiently bypasses the intermediate-to-bound state transition. The results demonstrate that inhibitor size and flexibility impact the binding mechanism, which could potentially modulate inhibitor residence time.

biophysics↗

bioRxiv2022

Hidden Markov Modeling of Molecular Orientations and Structures from High-Speed Atomic Force Microscopy Time-Series Images

High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between adjacent frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is widely applicable in integrative modeling of conformational dynamics using real experimental HS-AFM data. Author summaryBiomolecules dynamically change their structures in cells and perform various functions that are important for life. It is difficult to observe such dynamic structural changes using conventional experimental techniques, such as X-ray crystallography, that mainly observe static atomic structures. Alternative approach is the atomic force microscopy (AFM), a technique to measure the surface geometry of a sample molecule by scanning with an acute tip. Recently, by increasing the imaging rate of the AFM, it has become possible to observe the dynamics of biomolecules at work. However, the information measured with AFM is limited to the surface geometry of a molecule, so it is important to extract three-dimensional structural information from those data. In this study, we develop a method to estimate structural information with higher accuracy.

biophysics↗

bioRxiv2022

Structural Ramifications of Spike Protein D614G Mutation in SARS-CoV-2

A single mutation from aspartate to glycine at position 614 has dominated all circulating variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). D614G mutation induces structural changes in the Spike (S) protein that strengthen the virus infectivity. Here, we use molecular dynamics simulations to dissect the effects of mutation and 630-loop rigidification on wild-type structure. The introduction of mutation with ordered 630-loop induces structural changes toward S-G614 Cryo-EM structure. An ordered 630-loop weakens the stabilizing interactions of the anionic D614, suggesting its disorder in wild-type. The mutation allosterically alters the receptor binding domain (RBD) forming an asymmetric and mobile Down conformation, which facilitate Up transition. The loss of D614_K854 salt-bridge upon mutation, generally stabilize S-protein protomer, including the fusion peptide proximal region that mediates membrane fusion. Understanding of the molecular basis of D614G is crucial as it dominates in all variants of concern including Delta and Omicron.

biophysics↗

bioRxiv2022

Continuous Daily Head and Trunk Acceleration Recording for the Assessment of Bone Stimulation in Astronauts

The acceleration of the head and hip along the x-, y-, and z-axis of 14 healthy subjects was recorded during two sessions of 12 consecutive hours. The magnitude, frequency content, and root mean square of the acceleration signals were used to determine the type of physical activity (sitting, standing, walking, etc.) during normal daily life on Earth. The acceleration signal slope (jerk) was also calculated to assess whether these activities were sufficient to maintain bone mineral density. These measurements indicated that the changes in vertical acceleration experienced by our subjects during normal daily life were presumably sufficient to maintain bone mineral density. However, these changes might not be sufficient for postmenopausal women and astronauts during long-term exposure to weightlessness during spaceflight.

neuroscience↗

bioRxiv2021

Implementation of residue-level coarse-grained models in GENESIS for large-scale molecular dynamics simulations

Residue-level coarse-grained (CG) models have become one of the most popular tools in biomolecular simulations in the trade-off between modeling accuracy and computational efficiency. To investigate large-scale biological phenomena in molecular dynamics (MD) simulations with CG models, unified treatments of proteins and nucleic acids, as well as efficient parallel computations, are indispensable. In the GENESIS MD software, we implement several residue-level CG models, covering structure-based and context-based potentials for both well-folded biomolecules and intrinsically disordered regions. An amino acid residue in protein is represented as a single CG particle centered at the C atom position, while a nucleotide in RNA or DNA is modeled with three beads. Then, a single CG particle represents around ten heavy atoms in both proteins and nucleic acids. The input data in CG MD simulations are treated as GROMACS-style input files generated from a newly developed toolbox, GENESIS-CG-tool. To optimize the performance in CG MD simulations, we utilize multiple neighbor lists, each of which is attached to a different nonbonded interaction potential in the cell-linked list method. We found that random number generations for Gaussian distributions in the Langevin thermostat are one of the bottlenecks in CG MD simulations. Therefore, we parallelize the computations with message-passing-interface (MPI) to improve the performance on PC clusters or supercomputers. We simulate Herpes simplex virus (HSV) type 2 B-capsid and chromatin models containing more than 1,000 nucleosomes in GENESIS as examples of large-scale biomolecular simulations with residue-level CG models. This framework extends accessible spatial and temporal scales by multi-scale simulations to study biologically relevant phenomena, such as genome-scale chromatin folding or phase-separated membrane-less condensations. Author summaryMolecular dynamics (MD) simulations have been widely used to investigate biological phenomena that are difficult to study only with experiments. Since all-atom MD simulations of large biomolecular complexes are computationally expensive, coarse-grained (CG) models based on different approximations and interaction potentials have been developed so far. There are two practical issues in biological MD simulations with CG models. The first issue is the input file generations of highly heterogeneous systems. In contrast to well-established all-atom models, specific features are introduced in each CG model, making it difficult to generate input data for the systems containing different types of biomolecules. The second issue is how to improve the computational performance in CG MD simulations of heterogeneous biological systems. Here, we introduce a user-friendly toolbox to generate input files of residue-level CG models containing folded and disordered proteins, RNAs, and DNAs using a unified format and optimize the performance of CG MD simulations via efficient parallelization in GENESIS software. Our implementation will serve as a framework to develop novel CG models and investigate various biological phenomena in the cell.

biophysics↗

bioRxiv2021

The Inherent Flexibility of Receptor Binding Domains in SARS-CoV-2 Spike Protein

Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large conformational changes of its receptor binding domain (RBD) to enter the host cell, as the abundant structural studies suggest. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations without pre-defined reaction coordinates. The RBDA transition from Down to one-Up is supported by transient salt-bridges between RBDA and RBDC and by the glycan at N343B. Reduced interactions between RBDA and RBDB induce the RBDB motions toward two-Up. Glycan shielding for neutralizing antibodies is the weakest in one-Open. Cryptic pockets are revealed at the RBD interfaces in intermediate structures between Down and one-Up. The inherent flexibility in S-protein is, thus, essential for the structure transition and shall be considered for antiviral drug rational design or vaccine development.

biophysics↗

bioRxiv2021

Structural insight into Marburg virus nucleoprotein-RNA complex formation

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 [A]. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

microbiology↗

bioRxiv2021

Molecular basis for two stereoselective Diels-Alderases that produce decalin skeletons

Molecular chirality, discovered by Louis Pasteur in the middle of the 19th century1, is found in most primary and secondary metabolites. Particularly, the so-called natural products are rich in chiral centres2. The stereochemistry of natural products is strictly recognized in living organisms, and is thus closely related to their biological functions. The Diels-Alder (DA) reaction, which forms a six-membered ring with up to four chiral centres, is a fundamental practical reaction for C-C bond formation in synthetic chemistry3. Nature has also adopted this reaction to elaborate the complex structures of natural products using enzymes derived from various progenitor proteins4-7. Although enzymes catalysing the DA reaction, Diels-Alderases (DAases), have attracted increasing attention, little is known about the molecular mechanism by which they control the stereochemistry and perform catalysis. Here, we solved the X-ray crystal structures of a pair of decalin synthases, Fsa2 and Phm7, that catalyse intramolecular DA reactions to form enantiomeric decalin scaffolds during biosynthesis of the HIV-1 integrase inhibitor equisetin and its stereochemical opposite, phomasetin8,9. Based on the crystal structures, docking simulations followed by all-atom molecular dynamics simulations provided dynamic binding models demonstrating the folding of linear polyenoyl tetramic acid substrates in the binding pocket of these enzymes, explaining the stereoselectivity in the construction of decalin scaffolds. Site-directed mutagenesis studies verified the binding models and, in combination with density functional theory calculations, clarified how hydrophilic amino acid residues in the Phm7 pocket regulate and catalyse the stereoselective DA reaction. This study highlights the distinct molecular mechanisms of the enzymatic DA reaction and its stereoselectivity experimentally and computationally. We anticipate that clarified molecular mechanism herein provides not only the basic understanding how these important enzymes work but also the guiding principle to create artificial enzymes that produce designer bioactive molecules.

biochemistry↗

bioRxiv2020

GPI-anchoring is required for the proper transport and glycosylation of arabinogalactan protein precursor

Arabinogalactan proteins (AGPs) are extracellular proteoglycans with many O-linked glycan chains. Precursors to many AGPs contain a C-terminal signal for the addition of a glycosylphosphatidylinositol (GPI)-anchor, but the role of this modification has not been elucidated. NtAGP1, a tobacco precursor to AGP, comprises a signal peptide, an AGP-coding region, and a GPI-anchoring signal, and it is classified as a member of the classical AGP family. Using green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags and tobacco BY-2 cells as the host, we analysed the transport and modification of NtAGP1. The fusion protein of GFP or SPO and NtAGP1 expressed in BY-2 cells migrated as a large smear on SDS-polyacrylamide gel. A confocal microscopic analysis indicated that the GFP and NtAGP1 fusion protein localized to the plasma membrane (PM) and intracellular structures. Fractionation studies of microsomes indicated that most of the fusion protein of SPO and NtAGP1 (SPO-AGP) localized to the PM. In contrast, the expression of mutants without a GPI-anchoring signal yielded several forms. The largest forms migrating as large smears on the gel were secreted into the culture medium, whereas other forms were recovered in the endomembrane organelles. A comparison of the glycan structures of the microsomal SPO-AGP and the secreted mutant SPO-AGP without a GPI-anchoring signal using antibodies against AGP glycan epitopes indicated that the glycan structures of these proteins differ. These observations indicate that a GPI-anchoring signal is required for both the proper transport and glycosylation of the AGP precursor.

plant biology↗

bioRxiv2019

Implicit micelle model for membrane proteins using super-ellipsoid approximation

Surfactant micelles are often utilized as membrane mimetics for structure determination and functional analysis of membrane proteins. Although curved-surface effects of the micelle can perturb their structure, it is difficult to assess such effects and membrane mimetic artifacts by experimental and theoretical methods. Here, we propose an implicit micelle model (IMIC) to be used in molecular dynamics (MD) simulations of membrane proteins. IMIC is an extension of the IMM1 implicit membrane model by introducing a super-ellipsoid approximation to represent the curved-surface effects. Most of the parameters for IMIC are obtained from all-atom explicit solvent MD simulations of twelve membrane proteins in various micelles. In simulations of the HIV envelop protein gp41, M13 major coat protein gp8, and amyloid precursor protein (APP) dimer, curved-surface and compact hydrophobic-core effects are exhibited. The MD simulations with IMIC provide accurate structure predictions of membrane proteins in various micelle environments quickly with smaller computational cost than that necessary for explicit solvent/micelle model.

biophysics↗

bioRxiv2018

Chemo-mechanical Coupling in the Transport Cycle of a Type II ABC Transporter

AT P -binding cassette (ABC) transporters are integral membrane proteins that translocate a wide range of substrates across biological membranes, harnessing free energy from the binding and hydrolysis of ATP. To understand the mechanism of the inward- to outward-facing transition that could be achieved by tight regulation of ATPase activity through extensive conformational changes of the protein, we applied template-based iterative all-atom molecular dynamics (MD) simulation to the heme ABC transporter BhuUV-T. The simulations, together with biased MDs, predict two new conformations of the protein, namely, occluded (Occ) and outward-facing (OF) conformations. The comparison between the inward-facing crystal structure and the predicted two structures shows atomic details of the gating motions at the transmembrane helices and dimerization of the nucleotide-binding domains (NBDs). The MD simulations further reveal a novel role of the ABC signature motifs (LSGG[Q/E]) at the NBDs in decelerating ATPase activity in the Occ form through sporadic flipping of the side chains of the LSGG[Q/E] catalytic serine residues. The orientational changes are coupled to loose NBD dimerization in the Occ state, whereas they are blocked in the OF form where the NBDs are tightly dimerized. The chemo-mechanical coupling mechanism may apply to other types of ABC transporters having the conserved LSGG[Q/E] signature motifs.

biochemistry↗

bioRxiv2018

Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

The 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by {gamma}-secretase to form A{beta} peptide, which plays a critical role in the etiology of Alzheimers Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by {gamma}-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms {beta}-strands that may seed aggregation of A{beta} on the membrane surface, promoting amyloid formation. The N-terminus, which forms -helices that interact with the nicastrin domain of {gamma}-secretase. The C-terminus of C99 becomes more -helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become -helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

biophysics↗