DNA deformation by wildtype and mutant TBP.

<p>(A) DNA roll angle profile of TBP in the simulations of wildtype and mutant TBP proteins (the wildtype crystal structure 1cdw is shown as reference). The DNA sequence is shown as x-axis indicating that there are two kinks formed between a TA and an AG base step, respectively. (B) Time course of the roll angles for all base steps over the MD simulation. The two base steps exhibiting the largest kinks are highlighted by boxes at the y-axis. The vertical black line denotes the boundary between the two independent 250-ns MD simulations performed for each system, which are presented in a single panel. Colors of boxes match line colors of average roll angle plots of corresponding systems in (A). The type of mutation present in the individual systems is structurally depicted at the top of each panel.</p

Probing the role of intercalating protein sidechains for kink formation in DNA

<div><p>Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.</p></div

List of all simulated systems, the function of their protein part, types of intercalating residues and DNA selectivity at the kinked position.

<p>Residues in parenthesis are only partially intercalating.</p

DNA deformation by wildtype and mutant Sac7d.

<p>(A) DNA roll angle profile of average values calculated from the MD simulations. (B) Time course of the roll angles for all base steps over the MD simulation. The individual bases are plotted as y-axis starting with the 5’-end at the bottom. The lowest row in the diagram thus represents the time course of the roll angle between C1 and G2. The vertical black line denotes the boundary between the two independent 250-ns MD simulations performed for each system, which are presented in a single panel. Colors of boxes match line colors of average roll angle plots of corresponding systems in (A). (C) DNA roll angle profile of the roll angles found in the crystal structures of the wildtype and mutant Sac7d proteins. The PDB codes of the respective complexes are indicated. Wildtype and mutants are color coded as in (A).</p

Structural properties of the CcpA-DNA complex.

<p>(A) Structure of CcpA-DNA complex with intercalated residues highlighted in orange. The two subunits of CcpA are colored in blue and green, respectively, and the corepressor HPr is shown in gray. (B) Enlargement showing the kinked DNA base pair step and intercalating residues L56 and L56’. The kinked CG base step is also indicated. (C) DNA roll angle profile of CcpA in the simulations of wildtype and mutant CcpA proteins (the wildtype crystal structure 3oqm is shown as reference).</p

Flowchart of the approach to identify kinked DNA-protein complexes.

<p>Schematic presentation of the different steps taken to retrieve a set of 88 crystal structures of DNA-complexes for 15 different proteins from a total of 8617 structures available in the nucleic acid database NDB (see text for details of the procedure). The definition of the roll angle between two adjacent base pairs (red lines) is schematically depicted on the right.</p

Structural properties of the TFAM-DNA complex.

<p>(A) Structure of the TFAM-DNA complex. HMG-Box-2 is highlighted in color and HMG-Box-1 is shown in grey. (B) Enlargement showing the kinked CA base step (HMG-Box-2). The intercalating L140 as well as the partially inserting V124 and F128 are shown in stick presentation. (C) DNA roll angle profile of TFAM for the simulations of full-length and truncated systems containing only Box-2. The wildtype crystal structure (3tmm) is shown as reference. (D) DNA roll angle profiles for different TFAM Box-2 mutants.</p

Structure of the Sac7d-DNA complex.

<p>(A) Structure of the Sac7d-DNA complex with intercalated residues highlighted in orange. (B) Enlargement showing the kinked DNA base pair step and intercalating residues Val26 and Met29. The kinked CG base step is also indicated.</p

Probing the Structure of the Escherichia coli Periplasmic Proteins HdeA and YmgD by Molecular Dynamics Simulations

HdeA and YmgD are structurally homologous proteins in the periplasm of Escherichia coli. HdeA has been shown to represent an acid-activated chaperone, whereas the function of YmgD has not yet been characterized. We performed pH-titrating molecular dynamics simulations (pHtMD) to investigate the structural changes of both proteins and to assess whether YmgD may also exhibit an unfolding behavior similar to that of HdeA. The unfolding pathway of HdeA includes partially unfolded dimer structures, which represent a prerequisite for subsequent dissociation. In contrast to the coupled unfolding and dissociation of HdeA, YmgD displays dissociation of the folded subunits, and the subunits do not undergo significant unfolding even at low pH values. The differences in subunit stability between HdeA and YmgD may be explained by the structural features of helix D, which represents the starting point of unfolding in HdeA. In summary, the present study suggests that YmgD either is not an acid-activated chaperone or, at least, does not require unfolding for activation

Structure of the TBP-DNA complex.

<p>(A) Structure of TBP-DNA complex with intercalated residue highlighted in orange. (B) Enlargement showing one of the kinked DNA base pair steps (TA) and the intercalating residues Phe284 (loop) and Phe301 (sheet).</p