Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs

Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a red-shifted activation spectrum, cation selectivity, and large photocurrents while its slow closing kinetics impede some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests spectral and kinetic properties could be tuned with mutations and we identify ChRmine variants with two-fold increased and ten-fold decreased closing rates. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic manipulations.

Download Full-text

Cryo-EM Structures of Prestin and the Molecular Basis of Outer Hair Cell Electromotility

10.1101/2021.08.06.455374 ◽

2021 ◽

Author(s):

Navid Bavi ◽

Michael D Clark ◽

Gustavo F Contreras ◽

Rong Shen ◽

Bharat Reddy ◽

...

Keyword(s):

Outer Hair Cell ◽

Binding Pocket ◽

Structural Features ◽

Outer Hair Cells ◽

Anion Binding ◽

Structural Basis ◽

Inhibition Mechanism ◽

Core Domain ◽

The Core ◽

Voltage Dependent

The voltage-dependent motor protein, Prestin (SLC26A5) is responsible for the electromotive behavior of outer hair cells (OHCs). Here, we determined the structure of dolphin Prestin in complex with Cl- and the inhibitor Salicylate using single particle cryo-electron microscopy. These structures establish the specific structural features of mammalian Prestin and reveal small but significant differences with the transporter members of the SLC26 family of membrane proteins. Comparison with SLC26A9 point to conformational differences in the special relationship between the core and gate domains. Importantly, we highlight substantial alterations to the hydrophobic footprint of Prestin as it relates to the membrane, which point to a potential influence of Prestin on its surrounding lipid. The structure of Prestin bound to the inhibitor Salicylate confirms the nature of the anion binding pocket, formed by TM3 and TM10 in the Core domain and a set of anion coordinating residues which include Q97, F101, F137, S398 and R399. The presence of a well-defined density for Salycilate points to an inhibition mechanism based on competition for the anion-binding pocket of Prestin. These observations illuminate the structural basis of Prestin electromotility, a key component in the mammalian cochlear amplifier.

Download Full-text

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

10.1101/2021.08.15.456392 ◽

2021 ◽

Author(s):

Koichiro E. Kishi ◽

Yoon Seok Kim ◽

Masahiro Fukuda ◽

Tsukasa Kusakizako ◽

Elina Thadhani ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transmembrane Helix ◽

Action Spectrum ◽

Light Sensitivity ◽

Structural Basis ◽

Ion Pump ◽

Cryo Electron Microscopy ◽

Architectural Features ◽

Computational Analyses ◽

Insight Into

ChRmine, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics. 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 Å 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.

Download Full-text

Structural basis for binding mechanism of human serum albumin complexed with cyclic peptide dalbavancin

10.1101/2020.09.09.287375 ◽

2020 ◽

Author(s):

Sho Ito ◽

Akinobu Senoo ◽

Satoru Nagatoishi ◽

Masahito Ohue ◽

Masaki Yamamoto ◽

...

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Cyclic Peptides ◽

Cyclic Peptide ◽

Binding Pocket ◽

Structural Features ◽

Structural Basis ◽

Binding Mechanism ◽

Loop Region

ABSTRACTCyclic peptides, with unique structural features, have emerged as new candidates for drug discovery; their association with human serum albumin (HSA; long blood half-life), is crucial to improve drug delivery and avoid renal clearance. Here, we present the crystal structure of HSA complexed with dalbavancin, a clinically used cyclic peptide. SAXS and ITC experiments showed that the HSA-dalbavancin complex exists in a monomeric state; dalbavancin is only bound to the subdomain IA of HSA in solution. Structural analysis and MD simulation revealed that the swing of Phe70 and movement of the helix near dalbavancin were necessary for binding. The flip of Leu251 promoted the formation of the binding pocket with an induced-fit mechanism; moreover, the movement of the loop region including Glu60 increased the number of non-covalent interactions with HSA. These findings may support the development of new cyclic peptides for clinical use, particularly the elucidation of their binding mechanism to HSA.

Download Full-text

Solid-State Nuclear Magnetic Resonance Structural Study of the Retinal-Binding Pocket in Sodium Ion Pump Rhodopsin

Biochemistry ◽

10.1021/acs.biochem.6b00999 ◽

2017 ◽

Vol 56 (4) ◽

pp. 543-550 ◽

Cited By ~ 15

Author(s):

Arisu Shigeta ◽

Shota Ito ◽

Keiichi Inoue ◽

Takashi Okitsu ◽

Akimori Wada ◽

...

Keyword(s):

Nuclear Magnetic Resonance ◽

Solid State ◽

Magnetic Resonance ◽

Structural Study ◽

Binding Pocket ◽

Sodium Ion ◽

Ion Pump ◽

Retinal Binding Pocket ◽

Nuclear Magnetic

Download Full-text

Structural basis for potent and broad inhibition of HIV-1 RT by thiophene[3,2-d]pyrimidine non-nucleoside inhibitors

eLife ◽

10.7554/elife.36340 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 29

Author(s):

Yang Yang ◽

Dongwei Kang ◽

Laura A Nguyen ◽

Zachary B Smithline ◽

Christophe Pannecouque ◽

...

Keyword(s):

Reverse Transcriptase ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Structural Features ◽

Hiv Treatment ◽

Structural Basis ◽

Drug Resistant ◽

Hiv Therapy ◽

Anti Hiv ◽

Hiv 1

Rapid generation of drug-resistant mutations in HIV-1 reverse transcriptase (RT), a prime target for anti-HIV therapy, poses a major impediment to effective anti-HIV treatment. Our previous efforts have led to the development of two novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) with piperidine-substituted thiophene[3,2-d]pyrimidine scaffolds, compounds K-5a2 and 25a, which demonstrate highly potent anti-HIV-1 activities and improved resistance profiles compared with etravirine and rilpivirine, respectively. Here, we have determined the crystal structures of HIV-1 wild-type (WT) RT and seven RT variants bearing prevalent drug-resistant mutations in complex with K-5a2 or 25a at ~2 Å resolution. These high-resolution structures illustrate the molecular details of the extensive hydrophobic interactions and the network of main chain hydrogen bonds formed between the NNRTIs and the RT inhibitor-binding pocket, and provide valuable insights into the favorable structural features that can be employed for designing NNRTIs that are broadly active against drug-resistant HIV-1 variants.

Download Full-text

Structural Insights into Azole-based Inhibitors of Heme Oxygenase-1: Development of Selective Compounds for Therapeutic Applications

Current Medicinal Chemistry ◽

10.2174/0929867325666180606082512 ◽

2019 ◽

Vol 25 (42) ◽

pp. 5803-5821 ◽

Cited By ~ 3

Author(s):

Mona N. Rahman ◽

Dragic Vukomanovic ◽

Jason Z. Vlahakis ◽

Walter A. Szarek ◽

Kanji Nakatsu ◽

...

Keyword(s):

Heme Oxygenase ◽

Competitive Inhibition ◽

Cytochromes P450 ◽

Structural Similarity ◽

Binding Pocket ◽

Initial Study ◽

Structural Basis ◽

Heme Oxygenase 1 ◽

Design Strategies ◽

Inhibitor Binding

The development of isozyme-selective heme oxygenase (HO) inhibitors promises powerful pharmacological tools to elucidate the regulatory characteristics of the HO system. It is already known that HO has cytoprotective properties with a role in several disease states; thus, it is an enticing therapeutic target. Historically, the metalloporphyrins have been used as competitive HO inhibitors based on their structural similarity to the substrate, heme. However, heme’s important role in several other proteins (e.g. cytochromes P450, nitric oxide synthase), results in non-selectivity being an unfortunate side effect. Reports that azalanstat and other non-porphyrin molecules inhibited HO led to a multi-faceted effort over a decade ago to develop novel compounds as potent, selective inhibitors of HO. The result was the creation of the first generation of non-porphyrin based, non-competitive inhibitors with selectivity for HO, including a subset with isozyme selectivity for HO-1. Using X-ray crystallography, the structures of several complexes of HO-1 with novel inhibitors have been elucidated and provided insightful information regarding the salient features required for inhibitor binding. This included the structural basis for non-competitive inhibition, flexibility and adaptability of the inhibitor binding pocket, and multiple, potential interaction subsites, all of which can be exploited in future drug-design strategies. Notably, HO-1 inhibitors are of particular interest for the treatment of hyperbilirubinemia and certain types of cancer. Key features based on this initial study have already been used by others to discover additional potential HO-1 inhibitors. Moreover, studies have begun to use selected compounds and determine their effects in some disease models.

Download Full-text

Equilibrium and Kinetic Properties of Minerals

Mineralogical Magazine ◽

10.1180/002646198547909 ◽

1998 ◽

Vol 62 (5) ◽

pp. 581-583

Author(s):

Simon A. T. Redfern

Keyword(s):

Solid State ◽

Statistical Mechanics ◽

Structural Features ◽

Atomic Scale ◽

Kinetic Properties ◽

Computational Power ◽

Equilibrium Thermodynamics ◽

Bulk Properties ◽

Scale Characteristics ◽

Number Of Publications

How can the equilibrium and non-equilibrium thermodynamics of minerals be understood from their atomic-scale structural features? How can they be predicted, simply from models for the forces between atoms? Advances in analytical theory, statistical mechanics, experimental solid-state science, computational power, and the sophistication of a mineralogical approach that brings all of these together, means that these questions, once imponderable, are now realistically tractable. These questions have been exercising the minds of mineralogists over the last decade or so, and have motivated many developments in the science. Acting as way-markers along the path, there are a number of publications which have followed from meetings where these questions have been addressed. It is now twelve years since the publication of Microscopic to Macroscopic, an edition of Reviews in Mineralogy (Kieffer and Navrotsky, 1985) that sought to identify the fundamental controls on the bulk properties of minerals in terms of their atomic-scale characteristics.

Download Full-text

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

Nature Communications ◽

10.1038/s41467-020-20319-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qin Gong ◽

Kim Robinson ◽

Chenrui Xu ◽

Phuong Thao Huynh ◽

Kelvin Han Chung Chong ◽

...

Keyword(s):

Cell Death ◽

Inner Core ◽

Inflammatory Diseases ◽

Structural Features ◽

Structural Basis ◽

Cultured Human Cells ◽

Structural Insight ◽

Sensor Proteins ◽

Insight Into

AbstractNod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related sensor proteins NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, their mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to assemble distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core of oligomerized CARD surrounded by an outer ring of FIINDUPA. Biochemically, self-assembled NLRP1-CARD filaments are sufficient to drive ASC speck formation in cultured human cells—a process that is greatly enhanced by NLRP1-FIINDUPA which forms oligomers in vitro. The cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments, solved here at 3.7 Å, uncover unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide structural insight into the mechanisms of activation for human NLRP1 and CARD8 and reveal how highly specific signaling can be achieved by heterotypic CARD interactions within the inflammasome complexes.

Download Full-text

Structural basis for subtype-specific inhibition of the P2X7 receptor

eLife ◽

10.7554/elife.22153 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 94

Author(s):

Akira Karasawa ◽

Toshimitsu Kawate

Keyword(s):

P2x7 Receptor ◽

Hydrophobic Interactions ◽

Binding Pocket ◽

Drug Binding ◽

Structural Basis ◽

Atp Binding ◽

Allosteric Site ◽

Channel Opening ◽

A Site ◽

Novel Drug

The P2X7 receptor is a non-selective cation channel activated by extracellular adenosine triphosphate (ATP). Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. Here we present crystal structures of a mammalian P2X7 receptor complexed with five structurally-unrelated antagonists. Unexpectedly, these drugs all bind to an allosteric site distinct from the ATP-binding pocket in a groove formed between two neighboring subunits. This novel drug-binding pocket accommodates a diversity of small molecules mainly through hydrophobic interactions. Functional assays propose that these compounds allosterically prevent narrowing of the drug-binding pocket and the turret-like architecture during channel opening, which is consistent with a site of action distal to the ATP-binding pocket. These novel mechanistic insights will facilitate the development of P2X7-specific drugs for treating human diseases.

Download Full-text

Structural Basis for pH-Gating of the K+ Channel TWIK1 at the Selectivity Filter

10.1101/2021.11.09.467928 ◽

2021 ◽

Author(s):

Toby S Turney ◽

Vivian Li ◽

Stephen G Brohawn

Keyword(s):

Conformational Changes ◽

Pancreatic Beta Cells ◽

Low Ph ◽

Selectivity Filter ◽

Structural Basis ◽

K Channel ◽

Open Conformation ◽

Gating Mechanism ◽

Lipid Nanodiscs ◽

Pore Domain

TWIK1 is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a novel gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and show how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

Download Full-text