リゾホスファチジン酸受容体LPA6によるリガンド認識の構造基盤

学位の種別: 課程博士審査委員会委員 : （主査）東京大学准教授 小川 治夫, 東京大学教授 新井 洋由, 東京大学教授 嶋田 一夫, 東京大学教授 深田 吉孝, 東京大学教授 濡木 理University of Tokyo(東京大学

Time-resolved serial femtosecond crystallography reveals early structural changes in channelrhodopsin

X線自由電子レーザーを用いて、光照射によるチャネルロドプシンの構造変化の過程を捉えることに成功. 京都大学プレスリリース. 2021-03-26.Channelrhodopsins (ChRs) are microbial light-gated ion channels utilized in optogenetics to control neural activity with light . Light absorption causes retinal chromophore isomerization and subsequent protein conformational changes visualized as optically distinguished intermediates, coupled with channel opening and closing. However, the detailed molecular events underlying channel gating remain unknown. We performed time-resolved serial femtosecond crystallographic analyses of ChR by using an X-ray free electron laser, which revealed conformational changes following photoactivation. The isomerized retinal adopts a twisted conformation and shifts toward the putative internal proton donor residues, consequently inducing an outward shift of TM3, as well as a local deformation in TM7. These early conformational changes in the pore-forming helices should be the triggers that lead to opening of the ion conducting pore

Raw diffraction images of lysophosphatidic acid receptor LPA6

LPA6 is a class A G protein-coupled receptor which recognizes lysophosphatidic acid, a lipid mediator, as its ligand. The crystallization construct consists of zebrafish lysophosphatidic acid receptor LPA6 and T4 lysozyme fused within the intracellular loop 3 of LPA6. The crystals were obtained within the lipidic cubic phase. No synthetic chemical compounds were added for the crystallization. 397 small-wedge (4° or 6°/crystal) datasets collected from loop-harvested microcrystals using EIGER X 9M detector at a wavelength of 1 Å on BL32XU, SPring-8. The crystals belonged to space group P212121 with unit cell parameters a=55.9, b=65.0, c=160.7 Å. 241 datasets were merged at 3.2 Å resolution in the published result (Taniguchi et al. Nature 2017; PDB code: 5XSZ) using KAMO; see processing note https://github.com/keitaroyam/yamtbx/wiki/Processing-LPA6-data-(5XSZ) NOTE flatfield correction was not applied to the images and you need to apply it using the correction table saved in master.h5 files. master.h5 files were modified; see https://github.com/keitaroyam/yamtbx/blob/master/doc/eiger-en.md Most frames have lipid rings and some have ice rings

Translation dynamics in human cells visualized at high-resolution reveal cancer drug action

Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, yet their distribution in actively translating human cells remains elusive. Here, we optimized a cryo-electron tomography-based approach and resolved ribosome structures inside human cells with a local resolution of up to 2.5 angstroms. These structures revealed the distribution of functional states of the elongation cycle, a Z tRNA binding site and the dynamics of ribosome expansion segments. In addition, we visualized structures of Homoharringtonine, a drug for chronic myeloid leukemia treatment, within the active site of the ribosome and found that its binding reshaped the landscape of translation. Overall, our work demonstrates that structural dynamics and drug effects can be assessed at near-atomic detail within human cells

Atomistic design of microbial opsin-based blue-shifted optogenetics tools

Microbial opsins with a bound chromophore function as photosensitive ion transporters and have been employed in optogenetics for the optical control of neuronal activity. Molecular engineering has been utilized to create colour variants for the functional augmentation of optogenetics tools, but was limited by the complexity of the protein-chromophore interactions. Here we report the development of blue-shifted colour variants by rational design at atomic resolution, achieved through accurate hybrid molecular simulations, electrophysiology and X-ray crystallography. The molecular simulation models and the crystal structure reveal the precisely designed conformational changes of the chromophore induced by combinatory mutations that shrink its π-conjugated system which, together with electrostatic tuning, produce large blue shifts of the absorption spectra by maximally 100 nm, while maintaining photosensitive ion transport activities. The design principle we elaborate is applicable to other microbial opsins, and clarifies the underlying molecular mechanism of the blue-shifted action spectra of microbial opsins recently isolated from natural sources

Water-Containing Hydrogen-Bonding Network in the Active Center of Channelrhodopsin

Channelrhodopsin (ChR) functions as a light-gated ion channel in <i>Chlamydomonas reinhardtii.</i> Passive transport of cations by ChR is fundamentally different from the active transport by light-driven ion pumps such as archaerhodopsin, bacteriorhodopsin, and halorhodopsin. These microbial rhodopsins are important tools for optogenetics, where ChR is used to activate neurons by light, while the ion pumps are used for neural silencing. Ion-transport functions by these rhodopsins strongly depend on the specific hydrogen-bonding networks containing water near the retinal chromophore. In this work, we measured protein-bound water molecules in a chimeric ChR protein of ChR1 (helices A to E) and ChR2 (helices F and G) of <i>Chlamydomonas reinhardtii</i> using low-temperature FTIR spectroscopy at 77 K. We found that the active center of ChR possesses more water molecules (9 water vibrations) than those of other microbial (2–6 water vibrations) and animal (6–8 water vibrations) rhodopsins. We conclude that the protonated retinal Schiff base interacts with the counterion (Glu162) directly, without the intervening water molecule found in proton-pumping microbial rhodopsins. The present FTIR results and the recent X-ray structure of ChR reveal a unique hydrogen-bonding network around the active center of this light-gated ion channel

Nuclear pores safeguard the integrity of the nuclear envelope

Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes