Simulating Thioflavin T and Congo Red Binding to the Fibril Structure of Amyloid-ßß(1-42)

Binding modes for two amyloid-β(1-42) fibril tracers, namely Thioflavin T and Congo red, were identified using unbiased all-atom molecular dynamics simulations and binding free-energy computations. Both dyes bind to primarily hydrophobic grooves on the amyloid fibril surface, perpendicular to itsβ-strands. Binding affinities computed by the MM-GBSA method are in excellent agreement with experimental values and corroborate the proposed binding modes. The binding modes can guide the rational design of novel biomarkers for amyloid fibrils

Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects

Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specializedhepatocyte population and is essential for the maintenance of low, non‐toxic ammonia levelsin the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme inresponse to oxidative/nitrosative stress in a pH‐sensitive way. The underlying molecularmechanism as investigated by combined molecular simulations and in vitro experimentsindicates that tyrosine nitration can lead to a fully reversible and pH‐sensitive regulation ofprotein function. This approach was also used to understand the functional consequences ofseveral recently described point mutations of human GS with clinical relevance and to suggestan approach to restore impaired GS activity

Binding Modes of Thioflavin T and Congo Red to the Fibril Structure of Amyloid-β(1–42)

Binding modes for the amyloid-β(1–42) fibril fluorescent dyes thioflavin T and Congo red were predicted by molecular dynamics simulations and binding free energy calculations. Both probes bind on the fibril surface to primarily hydrophobic grooves, with their long axis oriented almost parallel to the fibril axis. The computed binding affinities are in agreement with experimental values. The binding modes also explain observables from previous structural studies and, thus, provide a starting point for the systematic search and design of novel molecules, which may improve in vitro diagnostics for Alzheimer's disease

Partially inserted nascent chain unzips the lateral gate of the Sec translocon

The Sec translocon provides the lipid bilayer entry for ribosome‐bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo‐electron microscopy‐based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α‐helices 2b, 7, and 8 tilt within the membrane core to “unzip” the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α‐helices of SecE subunit modulate the lateral gate conformation. Site‐specific cross‐linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology

Molecular Mechanisms of Glutamine Synthetase Mutations that Lead to Clinically Relevant Pathologies

Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the apo state of GS. Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically

Mechanism of fully-reversible, pH-sensitive inhibition of human glutamine synthetase by tyrosine nitration

Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reaction – and therefore GS – are indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding and configurational free energy computations that Y336 nitration hampers ATP binding, but only in the deprotonated and negatively-charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ~5.3. Thus, at physiological pH nitrated Y336 exists almost exclusively in the deprotonated and negatively-charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and pH 6, but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration

Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways

Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-μs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance