Biology subjects

Zacharias, M.

Publications and source records attributed to Zacharias, M..

21 records · Page 2Linked to original sources

bioRxiv2019

Phosphorylation of Ser111 in Rab8a modulates Rabin8 dependent activation by perturbation of side chain interaction networks

GTPases are key-players in cellular signaling processes. Phosphorylation of Rab proteins, which belong to the Ras superfamily of small GTPases regulating intracellular transport, has recently been implicated in the pathogenesis of Parkinson Disease (PD). For Rab8a, it was shown that serine 111 phosphorylation (pS111) is dependent on the protein kinase PINK1, and that mimicking the phosphorylation at S111 by a serine/glutamate substitution (S111E) impaired Rab8a activation by its cognate nucleotide exchange factor (GEF) Rabin8. Here, we performed comparative Molecular Dynamics and free energy simulations on Rab8a and Rab8a:Rabin8 complexes to elucidate the molecular details on how pS111 and S111E may influence the interaction with Rabin8. The simulations indicate that S111E and pS111 establish an intramolecular interaction with arginine 79 (R79). In the complex, this interaction persists, and therefore perturbs a favorable intermolecular salt-bridge contact between R79 in Rab8a and the acidic aspartate 187 (D187) in Rabin8. Binding free analysis reveals that S111E and pS111, as well as the mutation R79A, in Rab8a drastically reduce the binding affinity to Rabin8. Combining the R79A mutation with S111E or pS111, respectively, nearly diminishes Rab8a-Rabin8 binding. In vitro experiments confirm our computational results showing that the nucleotide exchange rates of the respective Rab8a mutants are decreased by >80% in the presence of Rabin8 compared to wild type. In addition to specific insights into how S111 phosphorylation of Rab8a can influence GEF-mediated activation, the simulations demonstrate how side chain modifications in general can allosterically influence the network of surface side chain interactions between binding partners.

biophysics↗

bioRxiv2019

Uncovering the Binding Mode of γ-Secretase Inhibitors

Knowledge of how transition state inhibitors bind to{gamma} -secretase is of major importance for the design of new Alzheimers disease therapies. Based on the known structure of{gamma} -secretase in complex with a fragment of the amyloid precursor protein we have generated a structural model of{gamma} -secretase in complex with the effective L-685,458 transition state inhibitor. The predicted binding mode is in excellent agreement with experimental data, mimicking all enzyme-substrate interactions at the active site and forming the relevant transition state geometry with the active site aspartate residues. In addition, we found that the stability of the complex is very likely also sensitive to the pH value. Comparative simulations on the binding of L-685,458 and the epimer L682,679 allowed us to explain the strongly reduced affinity of the epimer for{gamma} -secretase. The structural model could form a valuable basis for the design of new or modified{gamma} -secretase inhibitors.

biophysics↗

bioRxiv2018

Structural Modeling of Gamma-Secretase Aβn ComplexFormation and Substrate Processing

The intra-membrane aspartyl protease{gamma} -secretase (GSEC) cleaves single-span transmembrane helices including the C-terminal fragment of the amyloid precursor protein (APP). This substrate is initially cleaved at the {varepsilon}-site followed by successive processing (trimming) events mostly in steps of three amino acids. GSEC is responsible for the formation of N-terminal APP amyloid-{beta} (A {beta}) peptides of different length (e.g. A{beta}42) that can form aggregates involved in Alzheimers disease pathogenesis. The molecular mechanism of GSEC-APP substrate recognition is key for understanding how different peptide products are formed and could help in designing APP-selective modulators. Based on the known structure of apo GSEC and the APP-C99 fragment we have generated putative structural models of the initial binding in three different possible modes using extensive Molecular Dynamics (MD) simulations. The binding mode with the substrate helix located in a cleft between the transmembrane helices 2 and 3 of the presenilin subunit was identified as a most likely binding mode. Based on this arrangement the processing steps were investigated using restraint MD simulations to pull the scissile bond (for each processing step) into a transition like (cleavable) state. This allowed us to analyze in detail the motions and energetic contributions of participating residues. The structural model agrees qualitatively well with the influence of many mutations in GSEC and C99. It also explains the effects of inhibitors, cross-linking as well as spectroscopic data on GSEC substrate binding and can serve as working model for the future planning of structural and biochemical studies.

biophysics↗