Astashkin Roman
@mipt.ru
Moscow Institute of Physics and Technology
Scopus Publications
Scopus Publications
E. Podoliak, G. H. U. Lamm, E. Marin, A. V. Schellbach, D. A. Fedotov, A. Stetsenko, M. Asido, N. Maliar, G. Bourenkov, T. Balandin,et al.
Springer Science and Business Media LLC
AbstractLight-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.
Kirill Kovalev, Fedor Tsybrov, Alexey Alekseev, Vitaly Shevchenko, Dmytro Soloviov, Sergey Siletsky, Gleb Bourenkov, Michael Agthe, Marina Nikolova, David von Stetten,et al.
Springer Science and Business Media LLC
Sergey Bukhdruker, Tatsiana Varaksa, Philipp Orekhov, Irina Grabovec, Egor Marin, Ivan Kapranov, Kirill Kovalev, Roman Astashkin, Leonid Kaluzhskiy, Alexis Ivanov,et al.
International Union of Crystallography (IUCr)
New antitubercular drugs are vital due to the spread of resistant strains. Carbethoxyhexyl imidazole (CHImi) inhibits cytochrome P450 CYP124, which is a steroid-metabolizing enzyme that is important for the survival of Mycobacterium tuberculosis in macrophages. The available crystal structure of the CYP124–CHImi complex reveals two glycerol molecules in the active site. A 1.15 Å resolution crystal structure of the glycerol-free CYP124–CHimi complex reported here shows multiple conformations of CHImi and the CYP124 active site which were previously restricted by glycerol. Complementary molecular dynamics simulations show coherence of the ligand and enzyme conformations. Spectrophotometric titration confirmed the influence of glycerol on CHImi binding: the affinity decreases more than tenfold in glycerol-containing buffer. In addition, it also showed that glycerol has a similar effect on other azole and triazole CYP124 ligands. Together, these data show that glycerol may compromise structural–functional studies and impede rational drug-design campaigns.
R. Astashkin, K. Kovalev, S. Bukhdruker, S. Vaganova, A. Kuzmin, A. Alekseev, T. Balandin, D. Zabelskii, I. Gushchin, A. Royant,et al.
Springer Science and Business Media LLC
AbstractTransmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.
Igor Melnikov, Philipp Orekhov, Maksim Rulev, Kirill Kovalev, Roman Astashkin, Dmitriy Bratanov, Yury Ryzhykau, Taras Balandin, Sergei Bukhdruker, Ivan Okhrimenko,et al.
Springer Science and Business Media LLC
AbstractIn this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs – a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia.
V. Borshchevskiy, K. Kovalev, E. Round, R. Efremov, R. Astashkin, G. Bourenkov, D. Bratanov, T. Balandin, I. Chizhov, Christian Baeken,et al.
Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.
Alexander Kuzmin, Philipp Orekhov, Roman Astashkin, Valentin Gordeliy, and Ivan Gushchin
Wiley
Coronaviruses, especially SARS-CoV-2, present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43 and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate budding of the viral particles. The presented results may be helpful for better understanding of the function of coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.
Kirill Kovalev, Roman Astashkin, Valentin Gordeliy, and Vadim Cherezov
Springer US
D. Zabelskii, N. Dmitrieva, O. Volkov, V. Shevchenko, K. Kovalev, T. Balandin, D. Soloviov, R. Astashkin, Egor V. Zinovev, A. Alekseev,et al.
Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans . The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor. Zabelskii et al. present a structural and functional analysis of the lightdriven proton pump LR (Mac) from the fungus Leptosphaeria maculans. Their findings indicate that the archaeal ancestry of eukaryotic type 1 rhodopsins, and that the archaeal host of the proto-mitochondrial endosymbiont was capable of light-driven proton pumping.
Alina Remeeva, Vera V. Nazarenko, Kirill Kovalev, Ivan M. Goncharov, Anna Yudenko, Roman Astashkin, Valentin Gordeliy, and Ivan Gushchin
Wiley
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 å for Q148D to 1.63 å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins. This article is protected by copyright. All rights reserved.
Tatsiana Varaksa, Sergey Bukhdruker, Irina Grabovec, Egor Marin, Anton Kavaleuski, Anastasiia Gusach, Kirill Kovalev, Ivan Maslov, Aleksandra Luginina, Dmitrii Zabelskii,et al.
Elsevier BV
Mycobacterium tuberculosis (Mtb) infection is among top ten causes of death worldwide, and the number of drug-resistant strains is increasing. The direct interception of human immune signaling molecules by Mtb remains elusive, limiting drug discovery. Oxysterols and secosteroids regulate both innate and adaptive immune responses. Here we report a functional, structural, and bioinformatics study of Mtb enzymes initiating cholesterol catabolism and demonstrated their interrelation with human immunity. We show that these enzymes metabolize human immune oxysterol messengers. Rv2266 - the most potent among them - can also metabolize vitamin D3 (VD3) derivatives. High-resolution structures show common patterns of sterols binding and reveal a site for oxidative attack during catalysis. Finally, we designed a compound that binds and inhibits three studied proteins. The compound shows activity against Mtb H37Rv residing in macrophages. Our findings contribute to molecular understanding of suppression of immunity and suggest that Mtb has its own transformation system resembling the human phase I drug-metabolizing system.
Dmitrii Zabelskii, Alexey Alekseev, Kirill Kovalev, Vladan Rankovic, Taras Balandin, Dmytro Soloviov, Dmitry Bratanov, Ekaterina Savelyeva, Elizaveta Podolyak, Dmytro Volkov,et al.
Springer Science and Business Media LLC
Abstract Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability.
Kirill Kovalev, Roman Astashkin, Ivan Gushchin, Philipp Orekhov, Dmytro Volkov, Egor Zinovev, Egor Marin, Maksim Rulev, Alexey Alekseev, Antoine Royant,et al.
Springer Science and Business Media LLC
The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools. The Na + -pumping KR2 rhodopsin from Krokinobacter eikastus is a light-driven non-proton cation pump whose mechanism of pumping remains to be understood. Here authors solved crystal structures of the O-intermediate state of the pentameric form of KR2 and its D116N and H30A mutants, which sheds light on the mechanism of non-proton cation light-driven pumping.
C. Alleva, K. Kovalev, R. Astashkin, M. I. Berndt, C. Baeken, T. Balandin, V. Gordeliy, Ch. Fahlke, and J.-P. Machtens
American Association for the Advancement of Science (AAAS)
Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.
Nina Maliar, Kirill Kovalev, Christian Baeken, Taras Balandin, Roman Astashkin, Maksim Rulev, Alexey Alekseev, Nikolay Ilyinsky, Andrey Rogachev, Vladimir Chupin,et al.
MDPI AG
The light-driven sodium pump KR2, found in 2013 in the marine bacteria Krokinobacter eikastus, serves as a model protein for the studies of the sodium-pumping microbial rhodopsins (NaRs). KR2 possesses a unique NDQ (N112, D116, and Q123) set of the amino acid residues in the functionally relevant positions, named the NDQ motif. The N112 was shown to determine the Na+/H+ selectivity and pumping efficiency of the protein. Thus, N112A mutation converts KR2 into an outward proton pump. However, no structural data on the functional conversions of the light-driven sodium pumps are available at the moment. Here we present the crystal structure of the N112A mutant of KR2 in the ground state at the resolution of 2.4 Å. The structure revealed a minor deflection in the central part of the helix C and a double conformation of the L74 residue in the mutant. The organization of the retinal Schiff base and neighboring water molecules is preserved in the ground state of KR2-N112A. The presented data provide structural insights into the effects of the alterations of the characteristic NDQ motif of NaRs. Our findings also demonstrate that for the rational design of the KR2 variants with modified ion selectivity for optogenetic applications, the structures of the intermediate states of both the protein and its functional variants are required.
K. Kovalev, D. Volkov, R. Astashkin, A. Alekseev, I. Gushchin, J. M. Haro-Moreno, I. Chizhov, S. Siletsky, M. Mamedov, A. Rogachev,et al.
Proceedings of the National Academy of Sciences
BIOPHYSICS AND COMPUTATIONAL BIOLOGY Correction for “High-resolution structural insights into the heliorhodopsin family,” by K. Kovalev, D. Volkov, R. Astashkin, A. Alekseev, I. Gushchin, J. M. Haro-Moreno, I. Chizhov, S. Siletsky, M. Mamedov, A. Rogachev, T. Balandin, V. Borshchevskiy, A. Popov, G. Bourenkov, E. Bamberg, F. Rodriguez-Valera, G. Büldt, and V. Gordeliy, which was first published February 7, 2020; 10.1073/ pnas.1915888117 (Proc. Natl. Acad. Sci. U.S.A. 117, 4131–4141). The authors note that the affiliations d and h appeared incorrectly. The affiliation d should appear as “Research Center for Molecular Mechanisms of Aging and Age Related Diseases, Moscow Institute of Physics and Technology (National Research University), Dolgoprudny 141701, Russia.” The affiliation h should appear as “Belozersky Institute of Physical-Chemical Biology, Lomonosov Moscow State University, Moscow 119991, Russia.” The corrected author and affiliation lines appear below. The online version has been corrected.
K. Kovalev, D. Volkov, R. Astashkin, A. Alekseev, I. Gushchin, J. M. Haro-Moreno, I. Chizhov, S. Siletsky, M. Mamedov, A. Rogachev,et al.
Proceedings of the National Academy of Sciences
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.
Dmitry Bratanov, Kirill Kovalev, Jan-Philipp Machtens, Roman Astashkin, Igor Chizhov, Dmytro Soloviov, Dmytro Volkov, Vitaly Polovinkin, Dmitrii Zabelskii, Thomas Mager,et al.
Springer Science and Business Media LLC
Abstract Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.
Kirill Kovalev, Vitaly Polovinkin, Ivan Gushchin, Alexey Alekseev, Vitaly Shevchenko, Valentin Borshchevskiy, Roman Astashkin, Taras Balandin, Dmitry Bratanov, Svetlana Vaganova,et al.
American Association for the Advancement of Science (AAAS)
High resolution structures reveal the mechanisms of sodium, potassium, and proton pumping by a light-driven microbial rhodopsin.
Oleksandr Volkov, Kirill Kovalev, Vitaly Polovinkin, Valentin Borshchevskiy, Christian Bamann, Roman Astashkin, Egor Marin, Alexander Popov, Taras Balandin, Dieter Willbold,et al.
American Association for the Advancement of Science (AAAS)
The inner workings of an optogenetic tool Channelrhodopsins are membrane channel proteins whose gating is controlled by light. In their native setting, they allow green algae to move in response to light. Their expression in neurons allows precise control of neural activity, an approach known as optogenetics. Volkov et al. describe the high-resolution structure of channelrhodopsin 2, the most widely used optogenetics tool, as well as the structure of a mutant with a longer open-state lifetime (see the Perspective by Gerwert). Light activation perturbs an intricate hydrogen-bonding network to open the channel. The structures provide a basis for designing better optogenetic tools. Science, this issue p. 10.1126/science.eaan8862; see also p. 1000 Channelrhodopsin has an intricate hydrogen-bonding network that is perturbed by light activation, resulting in channel opening. INTRODUCTION Ion channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases. RATIONALE Despite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient. RESULTS We expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 Å, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible proton donor, to the RSB. The presence of the hydrogen bonds provides structural insights into how the DC gate controls ChR2 gating lifetime. CONCLUSION The determined structures of ChR2 and its C128T mutant present the molecular basis for the understanding of ChR functioning. They provide insights into mechanisms of channel opening and closing. A plausible scenario is that the disruption of the H-bonds between E123 and D253 and the Schiff base and the protonation of D253 upon retinal isomerization trigger rearrangements in the extended hydrogen-bonded networks, stabilizing the ECG and CG and also rearranging the H-bonding network in the cavities. Upon retinal isomerization, these two gates are opened and the network is broken. This leads to the reorientation of helix 2. Additional changes in helices 6 and 7 induced by the isomerization could help with opening the ICG and channel pore formation. General structure presentation of ChR2. (A) Four cavities and three gates forming the channel pore. (B) Extended hydrogen-bond network. The DC gate is shown in the red ellipse. The black arrows and gray horizontal lines show the putative ion pathway and position of hydrophobic/hydrophilic boundaries, respectively. The light-gated ion channel channelrhodopsin 2 (ChR2) from Chlamydomonas reinhardtii is a major optogenetic tool. Photon absorption starts a well-characterized photocycle, but the structural basis for the regulation of channel opening remains unclear. We present high-resolution structures of ChR2 and the C128T mutant, which has a markedly increased open-state lifetime. The structure reveals two cavities on the intracellular side and two cavities on the extracellular side. They are connected by extended hydrogen-bonding networks involving water molecules and side-chain residues. Central is the retinal Schiff base that controls and synchronizes three gates that separate the cavities. Separate from this network is the DC gate that comprises a water-mediated bond between C128 and D156 and interacts directly with the retinal Schiff base. Comparison with the C128T structure reveals a direct connection of the DC gate to the central gate and suggests how the gating mechanism is affected by subtle tuning of the Schiff base’s interactions.