Side-chain-induced changes in aminated chitosan: Insights from molecular dynamics simulations.

Chitosan is a functional polymer with diverse applications in biomedicine, agriculture, water treatment, and beyond. Via derivatization of pristine chitosan, its functionality can be tailored to desired applications, e.g. immobilization of biomolecules. Here, we performed molecular dynamics simulations of three aminated chitosan polymers, where one, two, and three long-distanced side chains have been incorporated. These polymers have been previously synthesized and their properties were investigated experimentally, however, the observed dependencies could not be fully explained on the molecular level. Here, we develop a computational protocol for the simulation of functionalized chitosan polymers and perform advanced analysis of their conformational states, intramolecular interactions, and water binding. We demonstrate that intra- and intermolecular forces, especially hydrogen bonds induced by polymer side chain modifications, modulate dihedral angle conformational states of the polymer backbone and interactions with water. We explain the role of the chemical composition of the functionalized chitosans in their tendency to collapse and reveal the key role of the protonation of the amino group near the polymer backbone on the reduction of polymer collapse. We demonstrate that specific binding of water molecules, especially the intermediate water, is more pronounced in the polymer exhibiting such an amino group.

A Strep-Tag Imprinted Polymer Platform for Heterogenous Bio(electro)catalysis.

Molecularly imprinted polymers (MIPs) are artificial receptors equipped with selective recognition sites for target molecules. One of the most promising strategies for protein MIPs relies on the exploitation of short surface-exposed protein fragments, termed epitopes, as templates to imprint binding sites in a polymer scaffold for a desired protein. However, the lack of high-resolution structural data of flexible surface-exposed regions challenges the selection of suitable epitopes. Here, we addressed this drawback by developing a polyscopoletin-based MIP that recognizes recombinant proteins via imprinting of the widely used Strep-tag II affinity peptide (Strep-MIP). Electrochemistry, surface-sensitive IR spectroscopy, and molecular dynamics simulations were employed to ensure an utmost control of the Strep-MIP electrosynthesis. The functionality of this novel platform was verified with two Strep-tagged enzymes: an O2-tolerant [NiFe]-hydrogenase, and an alkaline phosphatase. The enzymes preserved their biocatalytic activities after multiple utilization confirming the efficiency of Strep-MIP as a general biocompatible platform to confine recombinant proteins for exploitation in biotechnology.

Cation Dependence of Enniatin B/Membrane-Interactions Assessed Using Surface-Enhanced Infrared Absorption (SEIRA) Spectroscopy.

Enniatins are mycotoxins with well-known antibacterial, antifungal, antihelmintic and antiviral activity, which have recently come to attention as potential mitochondriotoxic anticancer agents. The cytotoxicity of enniatins is traced back to ionophoric properties, in which the cyclodepsipeptidic structure results in enniatin:cation-complexes of various stoichiometries proposed as membrane-active species. In this work, we employed a combination of surface-enhanced infrared absorption (SEIRA) spectroscopy, tethered bilayer lipid membranes (tBLMs) and density functional theory (DFT)-based computational spectroscopy to monitor the cation-dependence (Mz+=Na+, K+, Cs+, Li+, Mg2+, Ca2+) on the mechanism of enniatin B (EB) incorporation into membranes and identify the functionally relevant EBn : Mz+ complexes formed. We find that Na+ promotes a cooperative incorporation, modelled via an autocatalytic mechanism and mediated by a distorted 2 : 1-EB2 : Na+ complex. K+ (and Cs+) leads to a direct but less efficient insertion into membranes due to the adoption of "ideal" EB2 : K+ sandwich complexes. In contrast, the presence of Li+, Mg2+, and Ca2+ causes a (partial) extraction of EB from the membrane via the formation of "belted" 1 : 1-EB : Mz+ complexes, which screen the cationic charge less efficiently. Our results point to a relevance of the cation dependence for the transport into the malignant cells where the mitochondriotoxic anticancer activity is exerted.

Multi-structural molecular docking (MOD) combined with molecular dynamics reveal the structural requirements of designing broad-spectrum inhibitors of SARS-CoV-2 entry to host cells.

New variants of SARS-CoV-2 that can escape immune response continue to emerge. Consequently, there is an urgent demand to design small molecule therapeutics inhibiting viral entry to host cells to reduce infectivity rate. Despite numerous in silico and in situ studies, the structural requirement of designing viral-entry inhibitors effective against multiple variants of SARS-CoV-2 has yet to be described. Here we systematically screened the binding of various natural products (NPs) to six different SARS-CoV-2 receptor-binding domain (RBD) structures. We demonstrate that Multi-structural Molecular Docking (MOD) combined with molecular dynamics calculations allowed us to predict a vulnerable site of RBD and the structural requirement of ligands binding to this vulnerable site. We expect that our findings lay the foundation for in silico screening and identification of lead molecules to guide drug discovery into designing new broad-spectrum lead molecules to counter the threat of future variants of SARS-CoV-2.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

Ultrafast protein response in the Pfr state of Cph1 phytochrome.

Photoisomerization is a fundamental process in several classes of photoreceptors. Phytochromes sense red and far-red light in their Pr and Pfr states, respectively. Upon light absorption, these states react via individual photoreactions to the other state. Cph1 phytochrome shows a photoisomerization of its phycocyanobilin (PCB) chromophore in the Pfr state with a time constant of 0.7 ps. The dynamics of the PCB chromophore has been described, but whether or not the apoprotein exhibits an ultrafast response too, is not known. Here, we compare the photoreaction of 13C/15N labeled apoprotein with unlabeled apoprotein to unravel ultrafast apoprotein dynamics in Cph1. In the spectral range from 1750 to 1620 cm-1 we assigned several signals due to ultrafast apoprotein dynamics. A bleaching signal at 1724 cm-1 is tentatively assigned to deprotonation of a carboxylic acid, probably Asp207, and signals around 1670 cm-1 are assigned to amide I vibrations of the capping helix close to the chromophore. These signals remain after photoisomerization. The apoprotein dynamics appear upon photoexcitation or concomitant with chromophore isomerization. Thus, apoprotein dynamics occur prior to and after photoisomerization on an ultrafast time-scale. We discuss the origin of the ultrafast apoprotein response with the 'Coulomb hammer' mechanism, i.e. an impulsive change of electric field and Coulombic force around the chromophore upon excitation.

Structural and electronic properties of the active site of [ZnFe] SulE.

The function of the recently isolated sulerythrin (SulE) has been investigated using a combination of structural and electronic analyses based on quantum mechanical calculations. In the SulE structure of Fushinobu et al. (2003), isolated from a strictly aerobic archaeon, Sulfolobus tokadaii, a dioxygen-containing species was tentatively included at the active site during crystallographic refinement although the substrate specificity of SulE remains unclear. Studies have suggested that a structurally related enzyme, rubrerythrin, functions as a hydrogen peroxide reductase. Since SulE is a truncated version of rubrerythrin, the enzymes are hypothesized to function similarly. Hence, using available X-ray crystallography data (1.7 Å), we constructed various models of SulE containing a ZnII-Fe active site, differing in the nature of the substrate specificity (O2, H2O2), the oxidation level and the spin state of the iron ion, and the protonation states of the coordinating glutamate residues. Also, the substrate H2O2 is modeled in two possible configurations, differing in the orientation of the hydrogen atoms. Overall, the optimized geometries with an O2 substrate do not show good agreement with the experimentally resolved geometry. In contrast, excellent agreement between crystal structure arrangement and optimized geometries is achieved considering a H2O2 substrate and FeII in both spin states, when Glu92 is protonated. These results suggest that the dioxo species detected at the [ZnFe] active site of sulerythrin is H2O2, rather than an O2 molecule in agreement with experimental data indicating that only the diferrous oxidation state of the dimetal site in rubrerythrin reacts rapidly with H2O2. Based on our computations, we proposed a possible reaction pathway for substrate binding at the ZnFeII site of SulE with a H2O2 substrate. In this reaction pathway, Fe or another electron donor, such as NAD(P)H, catalyzes the reduction of H2O2 to water at the zinc-iron site.

Halogenation of tyrosine perturbs large-scale protein self-organization.

Protein halogenation is a common non-enzymatic post-translational modification contributing to aging, oxidative stress-related diseases and cancer. Here, we report a genetically encodable halogenation of tyrosine residues in a reconstituted prokaryotic filamentous cell-division protein (FtsZ) as a platform to elucidate the implications of halogenation that can be extrapolated to living systems of much higher complexity. We show how single halogenations can fine-tune protein structures and dynamics of FtsZ with subtle perturbations collectively amplified by the process of FtsZ self-organization. Based on experiments and theories, we have gained valuable insights into the mechanism of halogen influence. The bending of FtsZ structures occurs by affecting surface charges and internal domain distances and is reflected in the decline of GTPase activities by reducing GTP binding energy during polymerization. Our results point to a better understanding of the physiological and pathological effects of protein halogenation and may contribute to the development of potential diagnostic tools.

Infrared Spectroscopy Elucidates the Inhibitor Binding Sites in a Metal-Dependent Formate Dehydrogenase.

Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.

Photoinduced reaction mechanisms in prototypical and bathy phytochromes.

Phytochromes, found in plants, fungi, and bacteria, exploit light as a source of information to control physiological processes via photoswitching between two states of different physiological activity, i.e. a red-absorbing Pr and a far-red-absorbing Pfr state. Depending on the relative stability in the dark, bacterial phytochromes are divided into prototypical and bathy phytochromes, where the stable state is Pr and Pfr, respectively. In this work we studied representatives of these groups (prototypical Agp1 and bathy Agp2 from Agrobacterium fabrum) together with the bathy-like phytochrome XccBphP from Xanthomonas campestris by resonance Raman and IR difference spectroscopy. In all three phytochromes, the photoinduced conversions display the same mechanistic pattern as reflected by the chromophore structures in the various intermediate states. We also observed in each case the secondary structure transition of the tongue, which is presumably crucial for the function of phytochrome. The three phytochromes differ in details of the chromophore conformation in the various intermediates and the energetic barrier of their respective decay reactions. The specific protein environment in the chromophore pocket, which is most likely the origin for these small differences, also controls the proton transfer processes concomitant to the photoconversions. These proton translocations, which are tightly coupled to the structural transition of the tongue, presumably proceed via the same mechanism along the Pr → Pfr conversion whereas the reverse Pfr → Pr photoconversion includes different proton transfer pathways. Finally, classification of phytochromes in prototypical and bathy (or bathy-like) phytochromes is discussed in terms of molecular structure and mechanistic properties.

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.

Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator.

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel-iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level.

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light­absorbing) and Pfr (far-red light­absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/ß-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.

Tuning the Quantum Chemical Properties of Flavins via Modification at C8.

Flavins are central to countless enzymes but display different reactivities depending on their environments. This is understood to reflect modulation of the flavin electronic structure. To understand changes in orbital natures, energies, and correlation over the ring system, we begin by comparing seven flavin variants differing at C8, exploiting their different electronic spectra to validate quantum chemical calculations. Ground state calculations replicate a Hammett trend and reveal the significance of the flavin π-system. Comparison of higher-level theories establishes CC2 and ACD(2) as methods of choice for characterization of electronic transitions. Charge transfer character and electron correlation prove responsive to the identity of the substituent at C8. Indeed, bond length alternation analysis demonstrates extensive conjugation and delocalization from the C8 position throughout the ring system. Moreover, we succeed in replicating a particularly challenging UV/Vis spectrum by implementing hybrid QM/MM in explicit solvents. Our calculations reveal that the presence of nonbonding lone pairs correlates with the change in the UV/Vis spectrum observed when the 8-methyl is replaced by NH2, OH, or SH. Thus, our computations offer routes to understanding the spectra of flavins with different modifications. This is a first step toward understanding how the same is accomplished by different binding environments.

Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome.

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

Local Electric Field Changes during the Photoconversion of the Bathy Phytochrome Agp2.

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause-effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical-quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.

Beating Heart of Cytochrome c Oxidase: The Shared Proton of Heme a3 Propionates.

Cytochrome c oxidase (CcO) pumps protons from the N-side to the P-side and consumes electrons from the P-side of the mitochondrial membrane driven by energy gained from reduction of dioxygen to water. ATP synthesis uses the resulting proton gradient and electrostatic potential difference. Since the distance a proton travels through CcO is too large for a one-step transfer process, proton-loading sites (PLS) that can carry protons transiently are necessary. One specific pump-active PLS couples to the redox reaction, thus energizing the proton to move across the membrane against electric potential and proton gradient. The PLS should also prevent proton backflow. Therefore, the propionates of the two redox-active hemes in CcO were suggested as PLS candidates although, according to CcO crystal structures, none of the four propionates can be protonated on account of strong H-bonds. Here, we show that modeling the local structure around heme a3 propionates enhances significantly their capability of carrying a proton jointly. This was not possible for the propionates of heme a. The modeled structures are stable in molecular dynamics simulations (MDS) and are energetically similar to the crystal structure. Precise electrostatic energy computations of MDS data are used to estimate the pKA values of all titratable residues in CcO. For the modeled structures, the heme a3 propionates have pKA values high enough to host a proton transiently but not too high to fix the proton permanently. The change in pKA throughout the redox reaction is sufficient to push the proton to the P-side of the membrane and to provide the protons with the necessary amount of energy for ATP synthesis.

Hidden in Plain Sight: Natural Products of Commensal Microbiota as an Environmental Selection Pressure for the Rise of New Variants of SARS-CoV-2.

Since the emergence of SARS-CoV-2, little attention has been paid to the interplay between the interaction of virus and commensal microbiota. Here, we used molecular docking and dynamics simulations to study the interaction of some of the known metabolites and natural products (NPs) produced by commensal microbiota with the receptor binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. The results predict that NPs of commensal microbiota such as bile acids and non-ribosomal peptides (NRPs), of which some are siderophores, bind to the wild-type RBD and interfere with its binding to the ACE2 receptor. N501Y mutation, which is present in many of the emerging variants of the virus, abolishes the predicted binding pocket of bile acids and NRPs. Based on these findings, available experimental data showing that bile acids reduce the binding affinity of wild-type RBD to the ACE2 receptor, and the data suggesting that the respiratory tract microbiota affect viral infection we put forward the following proposal: mutations such as N501Y enable the RBD to bind to the ACE2 receptor more effectively in the presence of NPs produced by the respiratory tract bacteria thereby, increasing the infectivity rate of the virus. We hope our data stimulate future works to better understand the interactions of NPs produced by commensal microbiota with respiratory viruses like SARS-CoV-2.