scholarly journals The crystal structures of a chloride-pumping microbial rhodopsin and its proton-pumping mutant illuminate proton transfer determinants

2020 ◽  
Vol 295 (44) ◽  
pp. 14793-14804 ◽  
Author(s):  
Jessica E. Besaw ◽  
Wei-Lin Ou ◽  
Takefumi Morizumi ◽  
Bryan T. Eger ◽  
Juan D. Sanchez Vasquez ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Crystal Structures ◽  
Proton Pump ◽  
Chloride Transport ◽  
Chloride Ion ◽  
Proton Pumping ◽  
Single Amino Acid ◽  
Target Cells ◽  
Proton Pumps ◽  
Transport Pathway

Microbial rhodopsins are versatile and ubiquitous retinal-binding proteins that function as light-driven ion pumps, light-gated ion channels, and photosensors, with potential utility as optogenetic tools for altering membrane potential in target cells. Insights from crystal structures have been central for understanding proton, sodium, and chloride transport mechanisms of microbial rhodopsins. Two of three known groups of anion pumps, the archaeal halorhodopsins (HRs) and bacterial chloride-pumping rhodopsins, have been structurally characterized. Here we report the structure of a representative of a recently discovered third group consisting of cyanobacterial chloride and sulfate ion-pumping rhodopsins, the Mastigocladopsis repens rhodopsin (MastR). Chloride-pumping MastR contains in its ion transport pathway a unique Thr-Ser-Asp (TSD) motif, which is involved in the binding of a chloride ion. The structure reveals that the chloride-binding mode is more similar to HRs than chloride-pumping rhodopsins, but the overall structure most closely resembles bacteriorhodopsin (BR), an archaeal proton pump. The MastR structure shows a trimer arrangement reminiscent of BR-like proton pumps and shows features at the extracellular side more similar to BR than the other chloride pumps. We further solved the structure of the MastR-T74D mutant, which contains a single amino acid replacement in the TSD motif. We provide insights into why this point mutation can convert the MastR chloride pump into a proton pump but cannot in HRs. Our study points at the importance of precise coordination and exact location of the water molecule in the active center of proton pumps, which serves as a bridge for the key proton transfer.

scholarly journals Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

2015 ◽  
Vol 197 (16) ◽  
pp. 2704-2712 ◽  
Author(s):  
J. L. Keffer ◽  
M. W. Hahn ◽  
J. A. Maresca
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
High Performance ◽  
Proton Pumping ◽  
Host Cells ◽  
Metagenomic Data ◽  
Proton Pumps ◽  
Content Type ◽  
Knowing That ◽  
Light Capture

ABSTRACTRhodopsin-encoding microorganisms are common in many environments. However, knowing that rhodopsin genes are present provides little insight into how the host cells utilize light. The genome of the freshwater actinobacteriumRhodoluna lacicolaencodes a rhodopsin of the uncharacterized actinorhodopsin family. We hypothesized that actinorhodopsin was a light-activated proton pump and confirmed this by heterologously expressingR. lacicolaactinorhodopsin in retinal-producingEscherichia coli. However, cultures ofR. lacicoladid not pump protons, even though actinorhodopsin mRNA and protein were both detected. Proton pumping inR. lacicolawas induced by providing exogenous retinal, suggesting that the cells lacked the retinal cofactor. We used high-performance liquid chromatography (HPLC) and oxidation of accessory pigments to confirm thatR. lacicoladoes not synthesize retinal. These results suggest that in some organisms, the actinorhodopsin gene is constitutively expressed, but rhodopsin-based light capture may require cofactors obtained from the environment.IMPORTANCEUp to 70% of microbial genomes in some environments are predicted to encode rhodopsins. Because most microbial rhodopsins are light-activated proton pumps, the prevalence of this gene suggests that in some environments, most microorganisms respond to or utilize light energy. Actinorhodopsins were discovered in an analysis of freshwater metagenomic data and subsequently identified in freshwater actinobacterial cultures. We hypothesized that actinorhodopsin from the freshwater actinobacteriumRhodoluna lacicolawas a light-activated proton pump and confirmed this by expressing actinorhodopsin in retinal-producingEscherichia coli. Proton pumping inR. lacicolawas induced only after both light and retinal were provided, suggesting that the cells lacked the retinal cofactor. These results indicate that photoheterotrophy in this organism and others may require cofactors obtained from the environment.

scholarly journals Targeting the fungal plasma membrane proton pump.

1995 ◽  
Vol 42 (4) ◽  
pp. 481-496 ◽  
Author(s):  
B C Monk ◽  
A B Mason ◽  
T B Kardos ◽  
D S Perlin
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Proton Pump ◽  
Fungal Pathogens ◽  
Mutational Analysis ◽  
Proton Pumping ◽  
Proton Pumps ◽  
Essential Enzyme ◽  
Altered Cell ◽  
P Type

The need for new mechanistic classes of broad spectrum antifungal agents has prompted development of the membrane sector and ectodomain of the plasma membrane proton pumping ATPase as an antifungal target. The fungal proton pump is a highly abundant, essential enzyme in Saccharomyces cerevisiae. It belongs to the family of P-type ATPases, a class of enzymes that includes the Na+,K(+)-ATPase and the gastric H+,K(+)-ATPase. These enzymes are cell surface therapeutic targets for the cardiac glycosides and several anti-ulcer drugs, respectively. The effects of acid-activated omeprazole show that extensive inhibition of the S. cerevisiae ATPase is fungicidal. Fungal proton pumps possess elements within their transmembrane loops that distinguish them from other P-type ATPases. These loops, such as the conformationally sensitive transmembrane loop 1+2, can attenuate the activity of the enzyme. Expression in S. cerevisiae of fully functional chimeric ATPases that contain a foreign target comprising transmembrane loops 1+2 and/or 3+4 from the fungal pathogen Candida albicans suggests that these loops operate as a domain. The chimera containing C. albicans transmembrane loops 1+2 and 3+4 provides a prototype for mutational analysis of the target region and the screening of inhibitors directed against opportunistic fungal pathogens. Panels of mutants with modified ATPase regulation or with altered cell surface cysteine residues are also described. Information about the ATPase membrane sector and ectodomain has been integrated into a model of this region.

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

scholarly journals Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway

2017 ◽  
Vol 3 (8) ◽  
pp. e1700344 ◽  
Author(s):  
Tuo Zhang ◽  
Jian Liu ◽  
Matthias Fellner ◽  
Chi Zhang ◽  
Dexin Sui ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Zinc Transporter ◽  
Metal Center ◽  
Transport Pathway ◽  
Binuclear Metal

scholarly journals Decachlorocyclopentasilanes coordinated by pairs of chloride anions, with different cations, but the same solvent molecules

2017 ◽  
Vol 73 (12) ◽  
pp. 1903-1907 ◽  
Author(s):  
Maximilian Moxter ◽  
Julian Teichmann ◽  
Hans-Wolfram Lerner ◽  
Michael Bolte ◽  
Matthias Wagner
Keyword(s):  
Crystal Structures ◽  
General Position ◽  
Rotation Axis ◽  
Chloride Ion ◽  
The Other ◽  
Structural Motif ◽  
Solvent Molecules

We have determined the crystal structures of two decachlorocyclopentasilanes, namely bis(tetra-n-butylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C16H36N+·2Cl−·Si5Cl10·2CH2Cl2, (I), and bis(tetraethylammonium) dichloride decachlorocyclopentasilane dichloromethane disolvate, 2C8H20N+·2Cl−·Si5Cl10·2CH2Cl2, (II), both of which crystallize with discrete cations, anions, and solvent molecules. In (I), the complete decachlorocyclopentasilane ring is generated by a crystallographic twofold rotation axis. In (II), one cation is located on a general position and the other two are disordered about centres of inversion. These are the first structures featuring the structural motif of a five-membered cyclopentasilane ring coordinated from both sides by a chloride ion. The extended structures of (I) and (II) feature numerous C—H...Cl interactions. In (II), the N atoms are located on centres of inversion and as a result, the ethylene chains are disordered over equally occupied orientations.

scholarly journals Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

2021 ◽  
Vol 8 ◽  
Author(s):  
Sabine Panzer ◽  
Chong Zhang ◽  
Tilen Konte ◽  
Celine Bräuer ◽  
Anne Diemar ◽  
...  
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Aureobasidium Pullulans ◽  
Green Light ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Proton Pumps ◽  
C Terminus ◽  
Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

scholarly journals A computational study on how structure influences the optical properties in model crystal structures of amyloid fibrils

2017 ◽  
Vol 19 (5) ◽  
pp. 4030-4040 ◽  
Author(s):  
Luca Grisanti ◽  
Dorothea Pinotsi ◽  
Ralph Gebauer ◽  
Gabriele S. Kaminski Schierle ◽  
Ali A. Hassanali
Keyword(s):  
Optical Properties ◽  
Hydrogen Bonding ◽  
Proton Transfer ◽  
Crystal Structures ◽  
Amyloid Fibrils ◽  
Computational Study ◽  
Model Systems ◽  
Hydrogen Bonding Interactions ◽  
Model Crystal ◽  
Different Types

Different types of hydrogen bonding interactions that occur in amyloids model systems and molecular factors that control the susceptibility of the protons to undergo proton transfer and how this couples to the optical properties.

scholarly journals A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

2011 ◽  
Vol 437 (2) ◽  
pp. 269-278 ◽  
Author(s):  
José R. Pérez-Castiñeira ◽  
Agustín Hernández ◽  
Rocío Drake ◽  
Aurelio Serrano
Keyword(s):  
Fluorescent Dyes ◽  
Experimental System ◽  
Proton Pumping ◽  
Endocytic Pathway ◽  
Inorganic Pyrophosphatase ◽  
Proton Pumps ◽  
Specific Class ◽  
Ph Gradients ◽  
Atpase Subunit ◽  
Relative Contribution

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.

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