The Functionality of the DC Pair in a Rhodopsin Guanylyl Cyclase from Catenaria anguillulae.

Rhodopsin guanylyl cyclases (RGCs) belong to the class of enzymerhodopsins catalyzing the transition from GTP into the second messenger cGMP, whereas light-regulation of enzyme activity is mediated by a membrane-bound microbial rhodopsin domain, that holds the catalytic center inactive in the dark. Structural determinants for activation of the rhodopsin moiety eventually leading to catalytic activity are largely unknown. Here, we investigate the mechanistic role of the D283-C259 (DC) pair that is hydrogen bonded via a water molecule as a crucial functional motif in the homodimeric C. anguillulae RGC. Based on a structural model of the DC pair in the retinal binding pocket obtained by MD simulation, we analyzed formation and kinetics of early and late photocycle intermediates of the rhodopsin domain wild type and specific DC pair mutants by combined UV-Vis and FTIR spectroscopy at ambient and cryo-temperatures. By assigning specific infrared bands to S-H vibrations of C259 we are able to show that the DC pair residues are tightly coupled. We show that deprotonation of D283 occurs already in the inactive L state as a prerequisite for M state formation, whereas structural changes of C259 occur in the active M state and early cryo-trapped intermediates. We propose a comprehensive molecular model for formation of the M state that activates the catalytic moiety. It involves light induced changes in bond strength and hydrogen bonding of the DC pair residues from the early J state to the active M state and explains the retarding effect of C259 mutants.

Cascade Transformation of the Ansamycin Benzoquinone Core into Benzoxazole Influencing Anticancer Activity and Selectivity.

The metal-free cascade transformation of geldanamycin benzoquinone core is proposed at relatively mild conditions. This approach yields new benzoxazole ansamycin antibiotics and enables their functionalization in an atom-economic manner, irrespective of the type of amine used. The analysis of the heterocyclization course reveals the dependence of its rate on the nature of the para-substituent within the benzylamine moiety (EDG/EWG) and the strength of the base. The reduction of the ansamycin core enables an increase in anticancer potency and selectivity.

Modifications of geldanamycin via CuAAC altering affinity to chaperone protein Hsp90 and cytotoxicity.

Functionalization of alkyne (1) and azide (2) derivatives of geldanamycin (GDM) via dipolar cycloaddition CuAAC yielded 35 new congeners (3-37) with C(17)-triazole arms bearing caps of different nature (basic vs. acidic, hydrophilic vs. hydrophobic). Confrontation of biological data (anticancer activity vs. toxicity in normal cells) with lipophilicity (clogP), dissociation constants (Kd) of complexes with Hsp90 and binding modes to Hsp90 revealed SAR in specific subgroups of GDM derivatives. The most potent GDM congeners 14-16, bearing C(17)-triazole-benzyl-halogen arms exhibited the most optimal clogP values of 2.7-3.1 at favourable binding to Hsp90 (KdHsp90 at µM level). The anticancer activity of 14-16 (IC50 = 0.23-0.41 µM) is higher than those of GDM (IC50 = 0.58-0.64 µM) and actinomycin D (ActD, IC50 = 0.62-0.71 µM) in SKBR-3, SKOV-3 and PC-3 cell lines, with a comparable cytotoxicity in healthy cells. The relationship between structure and attractive anticancer potency (IC50 = 0.53-0.74 µM) is also observed for congeners with C(17)-triazole-saccharide or C(17)-triazole-unsaturated arms. In the former, the absolute configuration at C(4) (ᴅ-glucose vs. ᴅ-galactose) whereas in the latter the length of the unsaturated arm influences the cytotoxic effects due to different binding strength (Kd, ΔE) and modes with Hsp90. Among all triazole congeners of GDM that are biologically attractive and exhibit lower toxicity in normal cells than GDM and ActD, the derivative 22, bearing the C(17)-triazole-cinnamyl arm, shows the lowest Kd (Hsp90), optimal clogP = 2.82, the best pro-apoptotic properties in SKBR-3 and SKOV-3 and the best selectivity indices (SI). For the most potent GDM derivatives with C(17)-triazole arm, the docking studies have suggested the importance of the intermolecular stabilization between the arm and the D57 or Y61 of Hsp90.

Ultrafast proton-coupled isomerization in the phototransformation of phytochrome.

The biological function of phytochromes is triggered by an ultrafast photoisomerization of the tetrapyrrole chromophore biliverdin between two rings denoted C and D. The mechanism by which this process induces extended structural changes of the protein is unclear. Here we report ultrafast proton-coupled photoisomerization upon excitation of the parent state (Pfr) of bacteriophytochrome Agp2. Transient deprotonation of the chromophore's pyrrole ring D or ring C into a hydrogen-bonded water cluster, revealed by a broad continuum infrared band, is triggered by electronic excitation, coherent oscillations and the sudden electric-field change in the excited state. Subsequently, a dominant fraction of the excited population relaxes back to the Pfr state, while ~35% follows the forward reaction to the photoproduct. A combination of quantum mechanics/molecular mechanics calculations and ultrafast visible and infrared spectroscopies demonstrates how proton-coupled dynamics in the excited state of Pfr leads to a restructured hydrogen-bond environment of early Lumi-F, which is interpreted as a trigger for downstream protein structural changes.

Regioselective approach to colchiceine tropolone ring functionalization at C(9) and C(10) yielding new anticancer hybrid derivatives containing heterocyclic structural motifs.

The influence of base type, temperature, and solvent on regioselective C(9)/C(10) "click" modifications within the tropolone ring of colchiceine (2) is investigated. New ether derivatives of 2, bearing alkyne, azide, vinyl, or halide aryl groups enable assembly of the alkaloid part with heterocycles or important biomolecules such as saccharides, geldanamycin or AZT into hybrid scaffolds by dipolar cycloaddition (CuAAC) or Heck reaction. Compared to colchicine (1) or colchiceine (2), ether congeners, as e.g. 3e [IC50s(3e) ∼ 0.9 nM], show improved or similar anticancer effects, whereby the bulkiness of the substituents and the substitution pattern of the tropolone proved to be essential. Biological studies reveal that expanding the ether arms by terminal basic heterocycles as quinoline or pyridine, decreases the toxicity in HDF cells at high anticancer potency (IC50s ∼ 1-2 nM). Docking of ether and hybrid derivatives into the colchicine pocket of αGTP/ß tubulin dimers reveals a relationship between the favourable binding mode and the attractive anticancer potency.

The inner mechanics of rhodopsin guanylyl cyclase during cGMP-formation revealed by real-time FTIR spectroscopy.

Enzymerhodopsins represent a recently discovered class of rhodopsins which includes histidine kinase rhodopsin, rhodopsin phosphodiesterases, and rhodopsin guanylyl cyclases (RGCs). The regulatory influence of the rhodopsin domain on the enzyme activity is only partially understood and holds the key for a deeper understanding of intra-molecular signaling pathways. Here, we present a UV-Vis and FTIR study about the light-induced dynamics of a RGC from the fungus Catenaria anguillulae, which provides insights into the catalytic process. After the spectroscopic characterization of the late rhodopsin photoproducts, we analyzed truncated variants and revealed the involvement of the cytosolic N-terminus in the structural rearrangements upon photo-activation of the protein. We tracked the catalytic reaction of RGC and the free GC domain independently by UV-light induced release of GTP from the photolabile NPE-GTP substrate. Our results show substrate binding to the dark-adapted RGC and GC alike and reveal differences between the constructs attributable to the regulatory influence of the rhodopsin on the conformation of the binding pocket. By monitoring the phosphate rearrangement during cGMP and pyrophosphate formation in light-activated RGC, we were able to confirm the M state as the active state of the protein. The described setup and experimental design enable real-time monitoring of substrate turnover in light-activated enzymes on a molecular scale, thus opening the pathway to a deeper understanding of enzyme activity and protein-protein interactions.

Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2.

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield, i.e. the events of tongue refolding per absorbed photons, is lower by a factor of ca. two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators.

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1-7) and by quinuclidine motif (8), transformed into ammonium salts (9-13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC50s = 0.09-1.06 µM). Transformation of 8 into salts 9-13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC50∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC50∼2 µM to IC50∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8-13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.

Modulation of Light Energy Transfer from Chromophore to Protein in the Channelrhodopsin ReaChR.

The function of photoreceptors relies on efficient transfer of absorbed light energy from the chromophore to the protein to drive conformational changes that ultimately generate an output signal. In retinal-binding proteins, mainly two mechanisms exist to store the photon energy after photoisomerization: 1) conformational distortion of the prosthetic group retinal, and 2) charge separation between the protonated retinal Schiff base (RSBH+) and its counterion complex. Accordingly, energy transfer to the protein is achieved by chromophore relaxation and/or reduction of the charge separation in the RSBH+-counterion complex. Combining FTIR and UV-Vis spectroscopy along with molecular dynamics simulations, we show here for the widely used, red-activatable Volvox carteri channelrhodopsin-1 derivate ReaChR that energy storage and transfer into the protein depends on the protonation state of glutamic acid E163 (Ci1), one of the counterions of the RSBH+. Ci1 retains a pKa of 7.6 so that both its protonated and deprotonated forms equilibrate at physiological conditions. Protonation of Ci1 leads to a rigid hydrogen-bonding network in the active-site region. This stabilizes the distorted conformation of the retinal after photoactivation and decelerates energy transfer into the protein by impairing the release of the strain energy. In contrast, with deprotonated Ci1 or removal of the Ci1 glutamate side chain, the hydrogen-bonded system is less rigid, and energy transfer by chromophore relaxation is accelerated. Based on the hydrogen out-of-plane (HOOP) band decay kinetics, we determined the activation energy for these processes in dependence of the Ci1 protonation state.

Synthesis, structure and anticancer activity of new geldanamycin amine analogs containing C(17)- or C(20)- flexible and rigid arms as well as closed or open ansa-bridges.

The nucleophilic attack of amines at C(17) or C(17)/C(20) positions of geldanamycin's (GDM) benzoquinone, via initial 1,4-Michael conjugate addition mechanism, yield new analogs with closed or open ansa-bridges (1-31), respectively. X-ray structures of analogs 22 and 24 reveals an unexpected arrangement of the ansa-bridge in solid (conformer B), that is located between those of conformers A, prevailing in solution (trans-lactam), and C, crucial at binding to Hsp90 (cis-lactam). The structure of a new-type conformer B allows to better understand the molecular recognition mechanism between the GDM analogs and the target Hsp90. Combined analysis of: anticancer test results (SKBR-3, SKOV-3, PC-3, U-87, A-549) and those performed in normal cells (HDF), KD values and docking modes at Hsp90 as well as clogP parameters, reveals that the rigid C(17)-arm (piperidyl, cyclohexyl) with a H-bond acceptor as carbonyl group together with a lipophilicity clogP∼3 favor high potency of analogs, even up to IC50 ∼0.08 µM, at improved selectivity (SIHDF > 30), when compared to GDM. The most active 25 show higher anticancer potency than 17-AAG (in SKOV-3 and A-549) as well as reblastatin (in SKBR-3 and SKOV-3). Opening of the ansa-bridge within GDM analogs, at the best case, decreases activity (IC50∼2 µM) and toxicity in HDF cells (SIHDF∼2-3), relative to GDM.

Synthesis and Antibacterial Activity of New N-Alkylammonium and Carbonate-Triazole Derivatives within Desosamine of 14- and 15-Membered Lactone Macrolides.

Desosamines of azithromycin (AZM) and clarithromycin (CLA) were modified by N-alkylation or nucleophilic substitution at the carbonyl/CuAAC sequence. Biological studies revealed a higher antibacterial potency of quaternary N-alkylammonium bromides of CLA as compared to AZM. SAR studies of CLA salts, including biological, conformation and molecular-docking analysis, enriched by physicochemical parameters, showed the importance of less bulky and unsaturated substituent for an efficient docking mode at the ribosomal tunnel and good antibacterial potency against clinical and standard Streptococcus pneumoniae and Streptococcus pyogenes strains (MICs 0.25 or 0.5 µg/mL). These CLA salts also have an at least threefold lower cytotoxicity than reference antibiotics at comparable antibacterial activity against the S. pneumoniae clinical strain. Differences in antibacterial effects noted for AZM and CLA salts bearing less bulky N-substituents can be better understood when their binding modes in the ribosomal tunnel are considered rather than their common low lipophilicity and excellent water solubility.

Intramolecular Proton Transfer Controls Protein Structural Changes in Phytochrome.

Phytochromes are biological photoswitches that interconvert between two parent states (Pr and Pfr). The transformation is initiated by photoisomerization of the tetrapyrrole chromophore, followed by a sequence of chromophore and protein structural changes. In the last step, a phytochrome-specific peptide segment (tongue) undergoes a secondary structure change, which in prokaryotic phytochromes is associated with the (de)activation of the output module. The focus of this work is the Pfr-to-Pr photoconversion of the bathy bacteriophytochrome Agp2 in which Pfr is the thermodynamically stable state. Using spectroscopic techniques, we studied the structural and functional consequences of substituting Arg211, Tyr165, His278, and Phe192 close to the biliverdin (BV) chromophore. In Pfr, substitutions of these residues do not affect the BV structure. The characteristic Pfr properties of bathy phytochromes, including the protonated propionic side chain of ring C (propC) of BV, are preserved. However, replacing Arg211 or Tyr165 blocks the photoconversion in the Meta-F state, prior to the secondary structure transition of the tongue and without deprotonation of propC. The Meta-F state of these variants displays low photochemical activity, but electronic excitation causes ultrafast alterations of the hydrogen bond network surrounding the chromophore. In all variants studied here, thermal back conversion from the photoproducts to Pfr is decelerated but substitution of His278 or Phe192 is not critical for the Pfr-to-Pr photoconversion. These variants do not impair deprotonation of propC or the α-helix/ß-sheet transformation of the tongue during the Meta-F-to-Pr decay. Thus, we conclude that propC deprotonation is essential for restructuring of the tongue.

Féry Infrared Spectrometer for Single-Shot Analysis of Protein Dynamics.

Current submillisecond time-resolved broad-band infrared spectroscopy, one of the most frequently used techniques for studying structure-function relationships in life sciences, is typically limited to fast-cycling reactions that can be repeated thousands of times with high frequency. Notably, a majority of chemical and biological processes do not comply with this requirement. For example, the activation of vertebrate rhodopsin, a prototype of many protein receptors in biological organisms that mediate basic functions of life, including vision, smell, and taste, is irreversible. Here we present a dispersive single-shot Féry spectrometer setup that extends such spectroscopy to irreversible and slow-cycling systems by exploiting the unique properties of brilliant synchrotron infrared light combined with an advanced focal plane detector array embedded in a dispersive optical concept. We demonstrate our single-shot method on microbial actinorhodopsin with a slow photocycle and on vertebrate rhodopsin with irreversible activation.

Specific Interactions between Rifamycin Antibiotics and Water Influencing Ability To Overcome Natural Cell Barriers and the Range of Antibacterial Potency.

Rifamycins are a group of macrocyclic antibiotics mainly used for the treatment of various bacterial infections including tuberculosis. Spectroscopic studies of rifamycins evidence the formation of temperature- and solvent-dependent equilibria between A-, B-, and C-type conformers in solutions. The B- and C-type conformers of rifamycin antibiotics are exclusively formed in the presence of water molecules. A- and B-type conformers exhibit a hydrophilic and "open" ansa-bridge nature whereas the C-type conformer is more lipophilic due to the presence of a "closed" ansa-bridge structure. The involvement of the lactam moiety of the ansa-bridge in intramolecular H-bonds within rifapentine and rifampicin implicates the formation of a more hydrophilic A-type conformer. In contrast to rifampicin and rifapentine, for rifabutin and rifaximin, the "free" lactam group enhances conformational flexibility of the ansa-bridge, thereby enabling interconversion between A- and C-type conformers. In turn, an equilibrium between A- and C-type conformers for rifamycins improves their adaptation to the changing nature of bacteria cell membranes, especially those of Gram-negative strains and/or to efflux pump systems.

Role of the Propionic Side Chains for the Photoconversion of Bacterial Phytochromes.

Bacteriophytochromes harboring a biliverdin IXα (BV) chromophore undergo photoinduced reaction cascades to switch between physiologically inactive and active states. Employing vibrational spectroscopic and computational methods, we analyzed the role of propionic substituents of BV in the transformations between parent states Pr and Pfr in prototypical (Agp1) and bathy (Agp2) phytochromes from Agrobacterium fabrum. Both proteins form adducts with BV monoesters (BVM), esterified at propionic side chain B (PsB) or C (PsC), but in each case, only one monoester adduct is reactive. In the reactive Agp2-BVM-B complex (esterified at ring B), the Pfr dark state displays the structural properties characteristic of bathy phytochromes, including a protonated PsC. As in native Agp2, PsC is deprotonated in the final step of the Pfr phototransformation. However, the concomitant α-helix/ß-sheet secondary structure change of the tongue is blocked at the stage of unfolding of the coiled loop region. This finding and the shift of the tautomeric equilibrium of BVM toward the enol form are attributed to the drastic changes in the electrostatic potential. The calculations further suggest that deprotonation of PsC and the protonation state of His278 control the reactivity of the enol tautomer, thereby accounting for the extraordinarily slow thermal reversion. Although strong perturbations of the electrostatic potential are also found for Agp1-BVM, the consequences for the Pr-to-Pfr phototransformation are less severe. Specifically, the structural transition of the tongue is not impaired and thermal reversion is even accelerated. The different response of Agp1 and Agp2 to monoesterification of BV points to different photoconversion mechanisms.

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins.

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

Design of a light-gated proton channel based on the crystal structure of Coccomyxa rhodopsin.

The light-driven proton pump Coccomyxa subellipsoidea rhodopsin (CsR) provides-because of its high expression in heterologous host cells-an opportunity to study active proton transport under controlled electrochemical conditions. In this study, solving crystal structure of CsR at 2.0-Å resolution enabled us to identify distinct features of the membrane protein that determine ion transport directivity and voltage sensitivity. A specific hydrogen bond between the highly conserved Arg83 and the nearby nonconserved tyrosine (Tyr14) guided our structure-based transformation of CsR into an operational light-gated proton channel (CySeR) that could potentially be used in optogenetic assays. Time-resolved electrophysiological and spectroscopic measurements distinguished pump currents from channel currents in a single protein and emphasized the necessity of Arg83 mobility in CsR as a dynamic extracellular barrier to prevent passive conductance. Our findings reveal that molecular constraints that distinguish pump from channel currents are structurally more confined than was generally expected. This knowledge might enable the structure-based design of novel optogenetic tools, which derive from microbial pumps and are therefore ion specific.

Synthesis, docking and antibacterial studies of more potent amine and hydrazone rifamycin congeners than rifampicin.

New rifamycin congeners (1-33) with incorporated amine and hydrazone substituents leading to lipophilic and/or basic nature and altered rigidity of modified C(3) arm were synthesized and structurally characterized in detail. NMR spectroscopic studies at different temperatures indicate two types of structures of rifamycin congeners that are realized in solution: zwitterionic and non-ionic forms in dependence of the basicity of modified C(3) arm. The presence of rifamycin congeners in these two possible forms has a significant impact on the physico-chemical parameters such as lipophilicity (clogP) and water solubility and different binding mode of the C(3) arm of antibiotic at RNAP binding pocket (molecular target) leading to different antibacterial potency. The highest antibacterial potency against S. aureus (including MRSA and MLSB strains) and S. epidermidis strains, even higher than reference rifampicin (Rif) and rifaximin (Rifx) antibiotics, was found for rifamycin congeners bearing at the C(3) arm relatively rigid and basic substituents (bipiperidine and guanidine groups). These modifications provide favorable docking mode and excellent water solubility resulting in high potency (MICs 0.0078 µg/mL what gives ∼ 8.5 nM), irrespective whether rifamycin congener is a tertiary amine (15) or hydrazone (29). In turn, for a higher antibacterial potency of rifamycin congeners against E. faecalis strain (MICs 0.5 µg/mL that is 0.6 µM) as compared to Rif and Rifx, the most crucial factors are: bulkiness and the lipophilic character of the end of the C(3) rebuilt arm.

Tracking Pore Hydration in Channelrhodopsin by Site-Directed Infrared-Active Azido Probes.

In recent years, gating and transient ion-pathway formation in the light-gated channelrhodopsins (ChRs) have been intensively studied. Despite these efforts, a profound understanding of the mechanistic details is still lacking. To track structural changes concomitant with the formation and subsequent collapse of the ion-conducting pore, we site-specifically introduced the artificial polarity-sensing probe p-azido-l-phenylalanine (azF) into several ChRs by amber stop codon suppression. The frequently used optogenetic actuator ReaChR (red-activatable ChR) exhibited the best expression properties of the wild type and the azF mutants. By exploiting the unique infrared spectral absorption of azF [νas(N3) ∼ 2100 cm-1] and its sensitivity to polarity changes, we monitored hydration changes at various sites of the pore region and the inner gate by stationary and time-resolved infrared spectroscopy. Our data imply that channel closure coincides with a dehydration event occurring between the interface of the central and the inner gate. In contrast, the extracellular ion pathway seems to be hydrated in the open and closed states to similar extents. Mutagenesis of sites in the inner gate suggests that it acts as an intracellular entry funnel, whose architecture and composition modulate water influx and efflux within the channel pore. Our results highlight the potential of genetic code expansion technology combined with biophysical methods to investigate channel gating, particularly hydration dynamics at specific sites, with a so far unprecedented spatial resolution.