Korbinian Liebl and Martin Zacharias
The sequence dependent structure and exibility of the DNA double helix is of key importance for gene expression and DNA packing and it can be modulated by DNA modications. The presence of a C5'-methyl group in thymine or the frequent C5'-methylated-cytosine aects the DNA ne structure, however, the underlying mechanism and steric origins have remained largely unexplained. Employing Molecular Dynamics free energy simulations that allow switching on or o interactions with the methyl groups in several DNA sequences, we systematically identied the physical origin of the coupling between methyl groups and DNA backbone ne structure. Whereas methyl-solvent and methyl-nucleobase interactions were found to be of minor importance, the methyl group interaction with the 5'neighboring sugar was identied as main cause for in uencing the population of backbone substates. The sterical methyl sugar clash prevents the formation of unconventional stabilizing hydrogen bonds between nucleobase and backbone. The technique was also used to study the contribution of methyl groups to DNA exibility and served to explain why the presence of methyl sugar clashes in thymine and methyl-cytosine can result in an overall local increase of DNA exibility.
Atomic Resolution Insight into Sac7d Protein Binding to DNA and Associated Global Changes by Molecular Dynamics Simulations
Sac7d is a small, thermostable protein that induces large helical deformations in DNA upon association. Starting from multiple initial placements of the unbound Sac7d structure relative to a B-DNA oligonucleotide, molecular dynamics (MD) simulations were employed to directly follow several successful binding events at atomic resolution that resulted in structures in close agreement with the native complex geometry. The final native complex formed rapidly within tenths of nanoseconds and included simultaneous large-scale kinking, groove opening, twisting, and intercalation in the target DNA. The simulations indicate that the complex formation process involves initial non-native contacts that helped in reaching the final bound state, with residues intercalated at the center of the kinked DNA. It was also possible to identify several long-lived trapped intermediate states of the binding process and to follow sliding processes of Sac7d along the DNA minor groove.
Till Siebenmorgen and Martin Zacharias
The accurate prediction of protein-protein complex geometries is of major importance to ultimately model the complete interactome of interacting proteins in a cell. A major bottleneck is the realistic free energy evaluation of predicted docked structures. Typically, simple scoring functions applied to single-complex structures are employed that neglect conformational entropy and often solvent effects completely. The binding free energy of a predicted protein-protein complex can, however, be calculated using umbrella sampling (US) along a predefined dissociation/association coordinate of a complex. We employed atomistic US-molecular dynamics simulations including appropriate conformational and axial restraints and an implicit generalized Born solvent model to calculate binding free energies of a large set of docked decoys for 20 different complexes. Free energies associated with the restraints were calculated separately. In principle, the approach includes all energetic and entropic contributions to the binding process. The evaluation of docked complexes based on binding free energy calculation was in better agreement with experiment compared to a simple scoring based on energy minimization or MD refinement using exactly the same force field description. Even calculated absolute binding free energies of structures close to the native binding geometry showed a reasonable correlation to experiment. However, still for a number of complexes docked decoys of lower free energy than near-native geometries were found indicating inaccuracies in the force field or the implicit solvent model. Although time consuming the approach may open up a new route for realistic ranking of predicted geometries based on calculated free energy of binding.
Manuel Hitzenberger and Martin Zacharias
The intramembrane aspartyl protease γ-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 ϵ-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-β (Aβ) peptides of different length (e.g., Aβ42) that can form aggregates involved in Alzheimer's 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.