ABSTRACT
Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge. It requires true-atomic-resolution structures of all key proton-conducting states. Here we present a comprehensive function-structure study of a light-driven bacterial inward proton pump, xenorhodopsin, from Bacillus coahuilensis in all major proton-conducting states. The structures reveal that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. We demonstrate the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. The results might also be of interest for optogenetics since xenorhodopsins are the only alternative tools to fire neurons.
Subject(s)
Proton Pumps , Protons , Proton Pumps/chemistry , Ion TransportABSTRACT
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 1.15â 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.
Subject(s)
Cytochrome P-450 Enzyme System , Mycobacterium tuberculosis , Ligands , Models, Molecular , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/pharmacology , Antitubercular Agents , Crystallography, X-RayABSTRACT
Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins. In this chapter, we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases, also known as in meso approach. In particular, we describe in detail the methods of crystallization using lipidic cubic phase to grow both large crystals optimized for traditional crystallographic data collection and microcrystals for serial crystallography.
Subject(s)
Membrane Proteins , Rhodopsins, Microbial , Crystallization/methods , Crystallography, X-Ray , Lipids/chemistry , Membrane Proteins/chemistryABSTRACT
In 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.
Subject(s)
Anesthetics , Krypton , Argon/chemistry , Argon/pharmacology , Crystallography, X-Ray , Krypton/chemistry , Krypton/metabolism , LipidsABSTRACT
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.
Subject(s)
Proteins , Protons , Hydrogen BondingABSTRACT
Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the 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 the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.
Subject(s)
COVID-19 , Coronavirus Envelope Proteins/chemistry , SARS-CoV-2 , Humans , Protein Domains , Viral Envelope Proteins/chemistryABSTRACT
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.
Subject(s)
Proton Pumps/chemistry , Rhodopsin/chemistry , Ion Transport , Light , Phylogeny , Protein Domains , Rhodopsin/physiologyABSTRACT
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.
ABSTRACT
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.
Subject(s)
Energy Metabolism , Host-Pathogen Interactions , Mycobacterium tuberculosis/immunology , Tuberculosis/immunology , Tuberculosis/metabolism , 3-Hydroxysteroid Dehydrogenases/chemistry , 3-Hydroxysteroid Dehydrogenases/metabolism , Catalysis , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Enzyme Activation , Host-Pathogen Interactions/immunology , Humans , Immunity , Isoenzymes , Models, Molecular , Oxysterols/chemistry , Oxysterols/metabolism , Recombinant Proteins , Structure-Activity Relationship , Tuberculosis/microbiologyABSTRACT
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.
Subject(s)
Phytoplankton/virology , Rhodopsin/chemistry , Rhodopsin/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Animals , Calcium/metabolism , Cations , Cells, Cultured , Channelrhodopsins/metabolism , HEK293 Cells , Humans , Ion Channel Gating , Light , Neurons/metabolism , Phylogeny , Protein Conformation , Rats, Wistar , Rhodopsin/genetics , Structure-Activity Relationship , Viral Proteins/genetics , X-Ray DiffractionABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Flavobacteriaceae/metabolism , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium-Potassium-Exchanging ATPase/metabolism , Crystallography, X-Ray , Escherichia coli/metabolism , Molecular Dynamics Simulation , Protein Folding , Rhodopsin/chemistry , Rhodopsin/metabolism , Sodium/metabolism , X-Ray DiffractionABSTRACT
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.
Subject(s)
Phycodnaviridae/metabolism , Rhodopsins, Microbial/metabolism , Rhodopsins, Microbial/ultrastructure , Bacteriorhodopsins , Crystallography, X-Ray , Halobacterium salinarum , Ion Channel Gating , Ion Channels , Light , Molecular Dynamics Simulation , Protein Structure, Quaternary , Protein Structure, Tertiary , Rhodopsins, Microbial/physiologyABSTRACT
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.
Subject(s)
Rhodopsin/chemistry , Sodium-Potassium-Exchanging ATPase/chemistry , Sodium/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Hydrogen-Ion Concentration , Models, Molecular , Molecular Conformation , Mutation , Protein Binding , Protein Multimerization , Rhodopsin/genetics , Rhodopsin/metabolism , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Structure-Activity RelationshipABSTRACT
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.