Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

Rhodamine-Sensitized Two-Photon Activation of a Red Light-Absorbing BODIPY Photocage.

Two-photon (2P) activatable probes are of high value in biological and medical chemistry since near infrared (NIR) light can penetrate deeply even in blood-perfused tissue and due to the intrinsic three-dimensional activation properties. Designing two-photon chromophores is challenging. However, the two-photon absorption qualities of a photocage can be improved with an intramolecular sensitizer, which transfers the absorbed light onto the cage. We herein present the synthesis and photophysical characterization of a 2P-sensitive uncaging dyad based on rhodamine 101 as donor fluorophore and a redshifted BODIPY as acceptor photocage. Liberation of p-nitroaniline (PNA) upon one-photon photolysis was confirmed by HPLC analysis. The photoreaction was found to be accompanied by a considerable change of the fluorescence properties of the chromophores. The possibility of a fluorescent read-out enabled the detection of two-photon induced uncaging by confocal fluorescence microscopy.

Inhibition of soluble epoxide hydrolase prevents diabetic retinopathy.

Diabetic retinopathy is an important cause of blindness in adults, and is characterized by progressive loss of vascular cells and slow dissolution of inter-vascular junctions, which result in vascular leakage and retinal oedema. Later stages of the disease are characterized by inflammatory cell infiltration, tissue destruction and neovascularization. Here we identify soluble epoxide hydrolase (sEH) as a key enzyme that initiates pericyte loss and breakdown of endothelial barrier function by generating the diol 19,20-dihydroxydocosapentaenoic acid, derived from docosahexaenoic acid. The expression of sEH and the accumulation of 19,20-dihydroxydocosapentaenoic acid were increased in diabetic mouse retinas and in the retinas and vitreous humour of patients with diabetes. Mechanistically, the diol targeted the cell membrane to alter the localization of cholesterol-binding proteins, and prevented the association of presenilin 1 with N-cadherin and VE-cadherin, thereby compromising pericyte-endothelial cell interactions and inter-endothelial cell junctions. Treating diabetic mice with a specific sEH inhibitor prevented the pericyte loss and vascular permeability that are characteristic of non-proliferative diabetic retinopathy. Conversely, overexpression of sEH in the retinal Müller glial cells of non-diabetic mice resulted in similar vessel abnormalities to those seen in diabetic mice with retinopathy. Thus, increased expression of sEH is a key determinant in the pathogenesis of diabetic retinopathy, and inhibition of sEH can prevent progression of the disease.

Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp-His-Trp triad revealed by DNP-enhanced MAS-NMR.

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp-His-Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.

How Photoswitchable Lipids Affect the Order and Dynamics of Lipid Bilayers and Embedded Proteins.

Altering the properties of phospholipid membranes by light is an attractive option for the noninvasive manipulation of membrane proteins and cellular functions. Lipids with an azobenzene group within their acyl chains such as AzoPC are suitable tools for manipulating lipid order and dynamics through a light-induced trans-to-cis isomerization. However, the action of these photoswitchable lipids at the atomic level is still poorly understood. Here, liposomes containing AzoPC, POPE, and POPG have been characterized by solid-state NMR through chemical shift and dipolar CH order parameter measurements. Upon UV-light illumination, an efficient trans-to-cis conversion can be achieved resulting in a localized reduction of the CH order parameter within the bulk lipid acyl chains. This effect is even more pronounced in liposomes containing the integral membrane protein E. coli diacylglycerol kinase. The protein responds to the light-induced trans-to-cis isomerization by a site-specific increase in the molecular dynamics as observed by altered cross peak intensities in NCA spectra. This study represents a proof-of-concept demonstration for the use of photoswitchable lipids to modulate membrane properties by light for inducing dynamic changes within an embedded membrane protein.

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.

The Desensitized Channelrhodopsin-2 Photointermediate Contains 13 -cis, 15 -syn†Retinal Schiff Base.

Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.

Light-Induced Uncaging for Time-Resolved Observations of Biochemical Reactions by MAS NMR Spectroscopy.

Light-induced activation of biomolecules by uncaging of photolabile protection groups has found many applications for triggering biochemical reactions with minimal perturbations directly within cells. Such an approach might also offer unique advantages for solid-state NMR experiments on membrane proteins for initiating reactions within or at the membrane directly within the closed MAS rotor. Herein, we demonstrate that the integral membrane protein E. coli diacylglycerol kinase (DgkA), which catalyzes the phosphorylation of diacylglycerol, can be controlled by light under MAS-NMR conditions. Uncaging of NPE-ATP or of lipid substrate NPE-DOG by in situ illumination triggers its enzymatic activity, which can be monitored by real-time 31 P-MAS NMR. This proof-of-concept illustrates that combining MAS-NMR with uncaging strategies and illumination methods offers new possibilities for controlling biochemical reactions at or within lipid bilayers.

The molecular basis of subtype selectivity of human kinin G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.

Light Dynamics of the Retinal-Disease-Relevant G90D Bovine Rhodopsin Mutant.

The RHO gene encodes the G-protein-coupled receptor (GPCR) rhodopsin. Numerous mutations associated with impaired visual cycle have been reported; the G90D mutation leads to a constitutively active mutant form of rhodopsin that causes CSNB disease. We report on the structural investigation of the retinal configuration and conformation in the binding pocket in the dark and light-activated state by solution and MAS-NMR spectroscopy. We found two long-lived dark states for the G90D mutant with the 11-cis retinal bound as Schiff base in both populations. The second minor population in the dark state is attributed to a slight shift in conformation of the covalently bound 11-cis retinal caused by the mutation-induced distortion on the salt bridge formation in the binding pocket. Time-resolved UV/Vis spectroscopy was used to monitor the functional dynamics of the G90D mutant rhodopsin for all relevant time scales of the photocycle. The G90D mutant retains its conformational heterogeneity during the photocycle.

The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes.

Dynamic structural transitions within the seven-transmembrane bundle represent the mechanism by which G-protein-coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist-, and arrestin-bound states of Y2R were prepared by cell-free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13 C-labeled. NMR-signal assignment was achieved by dynamic-nuclear-polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13 C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved "cysteine lock"-Trp11623.50 was identified.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.

Exploring Protein Structures by DNP-Enhanced Methyl Solid-State NMR Spectroscopy.

Although the rapid development of sensitivity-enhanced solid-state NMR (ssNMR) spectroscopy based on dynamic nuclear polarization (DNP) has enabled a broad range of novel applications in material and life sciences, further methodological improvements are needed to unleash the full potential of DNP-ssNMR. Here, a new methyl-based toolkit for exploring protein structures is presented, which combines signal-enhancement by DNP with heteronuclear Overhauser effect (hetNOE), carbon-carbon-spin diffusion (SD) and strategically designed isotope-labeling schemes. It is demonstrated that within this framework, methyl groups can serve as dynamic sensors for probing local molecular packing within proteins. Furthermore, they can be used as "NMR torches" to selectively enlighten their molecular environment, e.g., to selectively enhance the polarization of nuclei within residues of ligand-binding pockets. Finally, the use of 13C-13C spin diffusion enables probing carbon-carbon distances within the subnanometer range, which bridges the gap between conventional 13C-ssNMR methods and EPR spectroscopy. The applicability of these methods is directly shown on a large membrane protein, the light-driven proton pump green proteorhodopsin (GPR), which offers new insight into the functional mechanism of the early step of its photocycle.

Host-Guest Interactions between Candesartan and Its Prodrug Candesartan Cilexetil in Complex with 2-Hydroxypropyl-ß-cyclodextrin: On the Biological Potency for Angiotensin II Antagonism.

Renin-angiotensin aldosterone system inhibitors are for a long time extensively used for the treatment of cardiovascular and renal diseases. AT1 receptor blockers (ARBs or sartans) act as antihypertensive drugs by blocking the octapeptide hormone Angiotensin II to stimulate AT1 receptors. The antihypertensive drug candesartan (CAN) is the active metabolite of candesartan cilexetil (Atacand, CC). Complexes of candesartan and candesartan cilexetil with 2-hydroxylpropyl-ß-cyclodextrin (2-HP-ß-CD) were characterized using high-resolution electrospray ionization mass spectrometry and solid state 13C cross-polarization/magic angle spinning nuclear magnetic resonance (CP/MAS NMR) spectroscopy. The 13C CP/MAS results showed broad peaks especially in the aromatic region, thus confirming the strong interactions between cyclodextrin and drugs. This experimental evidence was in accordance with molecular dynamics simulations and quantum mechanical calculations. The synthesized and characterized complexes were evaluated biologically in vitro. It was shown that as a result of CAN's complexation, CAN exerts higher antagonistic activity than CC. Therefore, a formulation of CC with 2-HP-ß-CD is not indicated, while the formulation with CAN is promising and needs further investigation. This intriguing result is justified by the binding free energy calculations, which predicted efficient CC binding to 2-HP-ß-CD, and thus, the molecule's availability for release and action on the target is diminished. In contrast, CAN binding was not favored, and this may allow easy release for the drug to exert its bioactivity.

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.

Unexplored Nucleotide Binding Modes for the ABC Exporter MsbA.

The ATP-binding cassette (ABC) transporter MsbA is an ATP-driven lipid-A flippase. It belongs to the ABC protein superfamily whose members are characterized by conserved motifs in their nucleotide binding domains (NBDs), which are responsible for ATP hydrolysis. Recently, it was found that MsbA could catalyze a reverse adenylate kinase (rAK)-like reaction in addition to ATP hydrolysis. Both reactions are connected and mediated by the same conserved NBD domains. Here, the structural foundations underlying the nucleotide binding to MsbA were therefore explored using a concerted approach based on conventional- and DNP-enhanced solid-state NMR, pulsed-EPR, and MD simulations. MsbA reconstituted into lipid bilayers was trapped in various catalytic states corresponding to intermediates of the coupled ATPase-rAK mechanism. The analysis of nucleotide-binding dependent chemical shift changes, and the detection of through-space contacts between bound nucleotides and MsbA within these states provides evidence for an additional nucleotide-binding site in close proximity to the Q-loop and the His-Switch. By replacing Mg2+ with Mn2+ and employing pulsed EPR spectroscopy, evidence is provided that this newly found nucleotide binding site does not interfere with the coordination of the required metal ion. Molecular dynamic (MD) simulations of nucleotide and metal binding required for the coupled ATPase-rAK mechanism have been used to corroborate these experimental findings and provide additional insight into nucleotide location, orientation, and possible binding modes.

The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR.

MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarization (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy.

Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to (15)N-labeled channelrhodopsin-2 carrying 14,15-(13)C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early [Formula: see text] K-like state, the slowly decaying late intermediate [Formula: see text], and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.

Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins.

Three all-trans retinals containing multiple 13 C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsin and channelrhodopsin. The synthetic approaches allowed specific introduction of 13 C labels in ring substituents and at different positions in the polyene chain to probe structural features such as ring orientation and interaction of the chromophore with the protein in the ground state and in photointermediates. [10-18-13 C9 ]-All-trans-retinal (1b), [12,15-13 C2 ]-all-trans-retinal (1c), and [14,15-13 C2 ]-all-trans-retinal (1d) were synthesized in in 12, 8, and 7 linear steps from ethyl 2-oxocyclohexanecarboxylate (5) or ß-ionone (4), respectively.

Overcoming Volume Selectivity of Dipolar Recoupling in Biological Solid-State NMR Spectroscopy.

Dipolar recoupling in solid-state NMR is an essential method for establishing correlations between nuclei that are close in space. In applications on protein samples, the traditional experiments like ramped and adiabatic DCP suffer from the fact that dipolar recoupling occurs only within a limited volume of the sample. This selection is dictated by the radiofrequency (rf) field inhomogeneity profile of the excitation solenoidal coil. We employ optimal control strategies to design dipolar recoupling sequences with substantially larger responsive volume and increased sensitivity. We show that it is essential to compensate for additional temporal modulations induced by sample rotation in a spatially inhomogeneous rf field. Such modulations interfere with the pulse sequence and decrease its performance. Using large-scale optimizations we developed pulse schemes for magnetization transfer from amide nitrogen to carbonyl (NCO) as well as aliphatic carbons (NCA). Our experiments yield a signal intensity increased by a factor of 1.5 and 2.0 for NCA and NCO transfers, respectively, compared to conventional ramped DCP sequences. Consistent results were obtained using several biological samples and NMR instruments.