Diversity of rhodopsin cyclases in zoospore-forming fungi.

Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum, consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.

Probing Factor Xa Protein-Ligand Interactions: Accurate Free Energy Calculations and Experimental Validations of Two Series of High-Affinity Ligands.

The accurate prediction of protein-ligand binding affinity belongs to one of the central goals in computer-based drug design. Molecular dynamics (MD)-based free energy calculations have become increasingly popular in this respect due to their accuracy and solid theoretical basis. Here, we present a combined study which encompasses experimental and computational studies on two series of factor Xa ligands, which enclose a broad chemical space including large modifications of the central scaffold. Using this integrated approach, we identified several new ligands with different heterocyclic scaffolds different from the previously identified indole-2-carboxamides that show superior or similar affinity. Furthermore, the so far underexplored terminal alkyne moiety proved to be a suitable non-classical bioisosteric replacement for the higher halogen-π aryl interactions. With this challenging example, we demonstrated the ability of the MD-based non-equilibrium free energy calculation approach for guiding crucial modifications in the lead optimization process, such as scaffold replacement and single-site modifications at molecular interaction hot spots.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

The mechanism behind enhanced reactivity of unsaturated phosphorus(v) electrophiles towards thiols.

Vinyl- and ethynyl phosphorus(v) electrophiles are a versatile class of thiol-reactive reagents suitable for cysteine-selective peptide and protein modifications, especially for the generation of antibody conjugates. Herein we investigated the reactivity of various P(v) reagents towards thiol addition. Complementing previous studies, we observed that the heteroatoms X (X = S, O, NH) as well as the vinyl- vs. ethynyl-substituent bound to phosphorus greatly influence the overall reactivity. These experimentally observed trends, as well as the high Z-selectivity for thiol additions to ethynyl derivatives, were further elucidated using DFT calculations. Hyperconjugation was a key means of stabilizing the intermediate generated upon the thiol addition, thus determining both the reactivity and stereoselectivity of unsaturated P(v) electrophiles. Specifically, the energetically low-lying σ antibonding orbital of the P-S bond more readily stabilizes the electron density from the lone pair (LP) of the generated carbanion, rendering the phosphonothiolates more reactive compared to the derivatives bearing oxygen and nitrogen. Our studies provide a detailed mechanistic picture for designing P(v)-based electrophiles with fine-tuned reactivity profiles.

Stereochemical Elucidation of Natural Products from Residual Chemical Shift Anisotropies in a Liquid Crystalline Phase.

Determination of the stereochemistry of organic molecules still represents one of the major obstacles in the structure elucidation procedure in drug discovery. Although the application of residual dipolar couplings (RDCs) has revolutionized this field, residual chemical shift anisotropies (RCSAs) which contain valuable structural information for nonprotonated carbons have only been scarcely employed so far. In this study, we present a simple but highly effective solution to extract RCSAs of the analytes in a liquid crystalline phase formed by AAKLVFF oligopeptides. This method does not require any special instruments, devices, or correction during postacquisition data analysis and thus can be easily applied in any chemistry laboratory. To illustrate the potential of this method, the relative configurations of four known natural products (1-4) belonging to different structural classes were confirmed. Moreover, we unambiguously elucidated the stereochemistry of spiroepicoccin A (5), a rare thiodiketopiperazine marine natural product whose configuration could not be assigned based on conventional NMR methods.

Combining free energy calculations with tailored enzyme activity assays to elucidate substrate binding of a phospho-lysine phosphatase.

Studying enzymes that are involved in the regulation of dynamic post-translational modifications (PTMs) is of key importance in proteomics research. Such investigations can be particularly challenging when the modification itself is intrinsically labile. In this article, we elucidate the enzymatic activity of Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) towards different O- and N-phosphorylated peptides by a combined experimental and computational approach. LHPP has been previously described to hydrolyze the phosphoramidate bonds in different small molecule substrates, including phosphorylated lysine (pLys). Taking the instability of the phosphoramidate bond into account, we conducted a carefully adjusted enzymatic assay with various pLys pentapeptides to confirm enzymatic phosphatase activity with LHPP. Molecular docking was employed to explore possible binding poses of the substrates in complex with the enzyme. Molecular dynamics based free energy calculations, which are unique in their accuracy and solid theoretical basis, were further applied to predict relative binding affinity of different substrates. Comparison of simulations with experiments clearly suggested a distinct binding motif of pLys peptides as well as a very narrow promiscuity of LHPP. We believe this integrated approach can be widely adopted to study the structure and interaction of poorly characterized enzyme-substrate complexes, in particular with synthetically challenging or labile substrates.

Characterization of H/D exchange in type 1 pili by proton-detected solid-state NMR and molecular dynamics simulations.

Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural dynamics. Deuterium back-exchange at labile sites of the perdeuterated, fully proton back-exchanged pili was conducted to investigate hydrogen/deuterium (H/D) exchange patterns of backbone amide protons in pre-assembled pili. We found distinct H/D exchange patterns in lateral and axial intermolecular interfaces in pili. Amide protons protected from H/D exchange in pili are mainly located in the core region of the monomeric subunit and in the lateral intermolecular interface, whereas the axial intermolecular interface and the exterior region of pili are highly exposed to H/D exchange. Additionally, we performed molecular dynamics simulations of the type 1 pilus rod and estimated the probability of H/D exchange based on hydrogen bond dynamics. The comparison of the experimental observables and simulation data provides insights into stability and mechanical properties of pili.

Comparison of the 3D structures of mouse and human α-synuclein fibrils by solid-state NMR and STEM.

Intra-neuronal aggregation of α-synuclein into fibrils is the molecular basis for α-synucleinopathies, such as Parkinson's disease. The atomic structure of human α-synuclein (hAS) fibrils was recently determined by Tuttle et al. using solid-state NMR (ssNMR). The previous study found that hAS fibrils are composed of a single protofilament. Here, we have investigated the structure of mouse α-synuclein (mAS) fibrils by STEM and isotope-dilution ssNMR experiments. We found that in contrast to hAS, mAS fibrils consist of two or even three protofilaments which are connected by rather weak interactions in between them. Although the number of protofilaments appears to be different between hAS and mAS, we found that they have a remarkably similar secondary structure and protofilament 3D structure as judged by secondary chemical shifts and intra-molecular distance restraints. We conclude that the two mutant sites between hAS and mAS (positions 53 and 87) in the fibril core region are crucial for determining the quaternary structure of α-synuclein fibrils.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR.

Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

Hybrid Structure of the Type 1 Pilus of Uropathogenic Escherichia coli.

Type 1 pili are filamentous protein assemblies on the surface of Gram-negative bacteria that mediate adhesion to host cells during the infection process. The molecular structure of type 1 pili remains elusive on the atomic scale owing to their insolubility and noncrystallinity. Herein we describe an approach for hybrid-structure determination that is based on data from solution-state NMR spectroscopy on the soluble subunit and solid-state NMR spectroscopy and STEM data on the assembled pilus. Our approach is based on iterative modeling driven by structural information extracted from different sources and provides a general tool to access pseudo atomic structures of protein assemblies with complex subunit folds. By using this methodology, we determined the local conformation of the FimA pilus subunit in the context of the assembled type 1 pilus, determined the exact helical pilus architecture, and elucidated the intermolecular interfaces contributing to pilus assembly and stability with atomic detail.