Biology subjects

Kusakizako, T.

Publications and source records attributed to Kusakizako, T..

8 recordsLinked to original sources

bioRxiv2025

Structural basis and physiological significance of non-canonical Gs coupling to the prototypical Gi-coupled melatonin MT1 receptor

G protein-coupled receptors (GPCRs) transduce extracellular stimuli into intracellular signals by coupling to various heterotrimeric G proteins. However, the rules governing G protein preference remain largely elusive. MT1 and MT2 are prototypical Gi/o-coupled GPCRs responding to melatonin, a hormone secreted in a circadian manner. We show here that MT1, but not MT2, couples also to Gs proteins in vitro and activates the Gs/cAMP pathway upon long-term melatonin exposure in vivo, mimicking physiological dawn conditions. We solved the cryo-electron microscopy structure of the melatonin-MT1-Gs complex at 3.0[A] resolution, which revealed a strikingly distinct binding mode compared to the MT1-Gi complex. The third intracellular loop of MT1 emerges as a key stabilizer for Gs coupling, a feature previously unrecognized. This is the first solved receptor-Gs complex of a primary Gi-coupled GPCRs, providing new structural and functional insights into G protein selectivity and circadian switch of G protein coupling.

molecular biology↗

bioRxiv2025

Structure-guided development of a potent human B0AT1 inhibitor effective in a mouse model of phenylketonuria

B0AT1 is a neutral amino acid transporter responsible for the (re)absorption in the intestine and kidney. Here, we developed small-molecule inhibitors of B0AT1 as a therapeutic strategy to a transient pocket, located [~]17 [A] away from the substrate-binding site and unique to the outward-open conformation, and stabilize an outward-occluded conformation that prevents the conformational transitions required for transport. Guided by structural insights, we optimized an initial inhibitor (Cinromide) to improve potency and cross-species activity, yielding compound 3, which inhibits both human and mouse B0AT1 with submicromolar IC_50 values. In a PKU mouse model (Pahenu2), oral administration of compound 3 increased urinary Phe excretion and significantly reduced plasma Phe. Our findings identify a druggable allosteric site in B0AT1, demonstrate its utility for achieving potent and selective inhibition in vivo, and establish allosteric blockade as a therapeutic approach for PKU and other SLC6-family transporters.

pharmacology and toxicology↗

bioRxiv2021

Structural insights into human excitatory amino acid transporter EAAT2

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity, that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. This is the first characterization of molecular features of EAAT2 and the selective inhibition mechanism, underlying structure-based drug design for EAAT2.

molecular biology↗

bioRxiv2021

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

ChRmine1, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin1, 2, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics1, 3-5. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 [A] resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

biophysics↗

bioRxiv2020

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

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 structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 [A] resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

biochemistry↗

bioRxiv2018

Cryo-EM structure of the volume-regulated anion channel LRRC8

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

molecular biology↗