Biology subjects

Yamashita, K.

Publications and source records attributed to Yamashita, K..

42 records · Page 3Linked to original sources

bioRxiv2020

Structural insights into the mechanism of rhodopsin phosphodiesterase

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase (Rh-GC) and photosensitive adenylyl cyclase (PAC). Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The 2.6 [A] resolution crystal structure of the transmembrane domain revealed a new topology of rhodopsin, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the transmembrane and PDE domain (2.1 [A]) with their connecting linkers. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

biophysics↗

bioRxiv2020

Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated ATP-release channels involved in neural functions including neurotransmission in gustation. Here we present the cryo-EM structures of killifish CALHM1, human CALHM2, and C. elegans CLHM-1 at resolutions of 2.66, 3.51, and 3.60 [A], respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state, and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonomer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the inter-subunit interactions at the transmembrane domain define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels. One Sentence SummaryCryo-EM structures reveal the ATP conduction and oligomeric assembly mechanisms of CALHM channels.

molecular biology↗

bioRxiv2019

Cryo-EM structures capturing the entire transport cycle of the P4-ATPase flippase

In eukaryotic membranes, P4-ATPases mediate the translocation of phospholipids from the outer to inner leaflet and maintain lipid asymmetry, which is critical for protein trafficking and signaling pathways. Here we report the cryo-EM structures of six distinct intermediates of the human ATP8A1-CDC50a hetero-complex, at 2.6-3.3 [A] resolutions, revealing the entire lipid translocation cycle of this P4-ATPase. ATP-dependent phosphorylation induces a large rotational movement of the actuator domain around the phosphorylation site, accompanied by lateral shifts of the first and second transmembrane helices, thereby allowing phosphatidylserine binding. The phospholipid head group passes through the hydrophilic cleft, while the acyl chain is exposed toward the lipid environment. These findings advance our understanding of the flippase mechanism and the disease-associated mutants of P4-ATPases.\n\nOne Sentence SummaryCryo-EM reveals lipid translocation by P4-type flippase.

biochemistry↗

bioRxiv2019

Cardiovascular effects of intravenous colforsin in normal and acute respiratory acidosis canine models: a dose-response study

AbstractIn acidosis, catecholamines are attenuated and higher doses are often required to improve cardiovascular function. Colforsin activates adenylate cyclase in cardiomyocytes without mediating the beta adrenoceptor. In this study, six beagles were administered either colforsin or dobutamine four times during eucapnia (partial pressure of arterial carbon dioxide 35-40 mm Hg; normal) and hypercapnia (ibid 90-110 mm Hg; acidosis) conditions. The latter was induced by carbon dioxide inhalation. Anesthesia was induced with propofol and maintained with isoflurane. Cardiovascular function was measured by thermodilution and a Swan-Ganz catheter. Cardiac output, heart rate, and systemic vascular resistance were determined at baseline and 60 min after 0.3 g/kg/min (low), 0.6 g/kg/min (middle), and 1.2 g/kg/min (high) colforsin administration. The median pH was 7.38 [range 7.34-7.42] and 7.04 [range 7.01-7.08] at baseline in the Normal and Acidosis conditions, respectively. Endogenous adrenaline and noradrenaline levels at baseline were significantly (P < 0.05) higher in the Acidosis than in the Normal condition. Colforsin induced cardiovascular effects similar to those caused by dobutamine. Colforsin increased cardiac output in the Normal condition (baseline: 198.8 mL/kg/min [range 119.6-240.9], low: 210.8 mL/kg/min [range 171.9-362.6], middle: 313.8 mL/kg/min [range 231.2-473.2], high: 441.4 mL/kg/min [range 373.9-509.3]; P < 0.001) and the Acidosis condition (baseline: 285.0 mL/kg/min [range 195.9-355.0], low: 297.4 mL/kg/min [213.3-340.6], middle: 336.3 mL/kg/min [291.3-414.5], high: 366.7 mL/kg/min [339.7-455.7] ml/kg/min; P < 0.001). Colforsin significantly increased heart rate (P < 0.05 in both conditions) and decreased systemic vascular resistance (P < 0.05 in both conditions) compared to values at baseline. Systemic vascular resistance was lower in the Acidosis than in the Normal condition (P < 0.001). Dobutamine increased pulmonary artery pressure, whereas colforsin did not. Colforsin offsets the effects of endogenous catecholamines and may not increase cardiac output during hypercapnia.

pharmacology and toxicology↗

bioRxiv2018

X-ray structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signaling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. The effectiveness of a highly ETB-selective endothelin analogue, IRL1620, has been investigated in clinical trials. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonists, endohelin-3 and IRL1620. The 2.0 [A]-resolution structure of the endothelin-3-bound receptor revealed that the disruption of water-mediated interactions between W6.48 and D2.50, which are highly conserved among class A GPCRs, is critical for receptor activation. These hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure, and a functional analysis revealed the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

molecular biology↗

bioRxiv2017

Structure of the triose-phosphate/phosphate translocator reveals the basis of substrate specificity

The triose-phosphate/phosphate translocator (TPT) catalyzes the strict 1:1 exchange of triose phosphate, 3-phosphoglycerate and inorganic phosphate across the chloroplast envelope, and plays crucial roles in photosynthesis. Despite rigorous studies for more than 40 years, the molecular mechanism of TPT is poorly understood due to the lack of structural information. Here we report crystal structures of TPT bound to two different substrates, 3-phosphoglycerate and inorganic phosphate, in occluded conformations. The structures reveal that TPT adopts a 10-transmembrane drug/metabolite transporter fold. Both substrates are bound within the same central pocket, where conserved lysine, arginine, and tyrosine residues recognize the shared phosphate group. A structural comparison with the outward-open conformation of the bacterial drug/metabolite transporter suggests a rocking-type motion of helix bundles, and molecular dynamics simulations support a model in which this helix rocking is tightly coupled to the substrate binding, to ensure strict 1:1 exchange. These results reveal the unique mechanism of sugar phosphate/phosphate exchange by TPT.

biochemistry↗