The profile of residual flexibility of p53 DBD, as indicated by root mean square fluctuation (RMSF).

<p>The RMSF for all concatenated MD conformers are shown in grey histogram. The RMSFs for extended and recessed L1 conformers are shown in magenta and cyan lines, respectively. Black and grey boxes represent alpha helices and beta sheets, respectively. L1, L2 and L3 are indicated as blue, green and red lines, respectively.</p

L1 and H2 are distant to each other in recessed L1 conformers.

<div><p>(A) The profile of the distances between residues in L1 and H2 that increase in recessed L1 conformers, across experimentally determined structures. </p> <p>(B) The profile of the distances indicated in (A) across MD conformers analyzed in this study. Horizontal dotted lines indicate the threshold for the distances in experimentally determined structures with recessed L1 conformations. Vertical dotted lines demarcate independent MD simulations started from four different starting structures, each with two copies of different initial velocity. </p> <p>(C-E) Reaction coordinates to characterize distinct L1 conformations, where recessed L1 conformational space is sampled by MD conformers (blue). Crystal and NMR structures are colored in yellow and magenta respectively. </p> <p>(F) Residues T118, K120, R280-R283 are shown as sticks. </p></div

Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures

<div><p>(<b>see</b> Figure 2). </p> <p>The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.</p></div

The proposed rescue mutants of L1 for the oncogenic R248Q, whose L3 experiences enhanced flexibility due to the mutation.

<p>In the L114R mutant, R114 forms hydrogen bonds with S116 of L1 and C124. In the G117R mutant, R117 forms a hydrogen bonds with A119. In the T118N mutant, N118 forms a hydrogen bond with R283. In each panel, L1, L2, L3 and DNA are colored in blue, green, red and pink, respectively.</p

Structure of p53 DNA binding domain.

<div><p>(A) The primary sequence of p53 comprising 393 amino acid residues was retrieved from Uniprot P04637 isoform 1. For p53 DNA binding domain, the secondary structure contents of alpha-helices (black) and beta-sheets (gray) are indicated on top of amino acid positions. The important loops in p53 DNA binding domain: loop 1, loop 2, loop 3 are colored in blue, green, red, respectively.</p> <p>(B) The structures of DNA-bound p53 DBD from 3Q05 chain A (dark grey) with recessed L1 and from 3Q05 chain B (light grey) with extended L1. DNA is shown as pink transparent surface and cartoon representations. The important loops in p53 DBD: loop 1, loop 2, loop 3 are colored in blue, green, red, respectively. The following residues are shown in sticks: K120 (cyan), S121 (magenta), V122 (yellow), R280 (light blue), R283 (purple).</p> <p>(C) The structure of p53 DBD colored according to spectrum. Secondary structures are labeled. The PDB 2OCJ chain A was used to generate this figure.</p> <p>(D) The same figure as (C) except that only loop 1, loop 2, and loop 3 are colored in blue, green, and red, respectively. The zinc ion is shown as an orange sphere. </p></div

Projection of experimentally determined conformers of p53 DNA binding domain (DBD) onto the principal planes defined by the significant principal components (PC1 - PC3).

<div><p>(A-C) Structures determined using X-ray crystallography and nuclear magnetic resonance are colored in yellow and magenta respectively. Structures crystallized with DNA are indicated with an ‘x’. The PDB codes of experimental structures used for our subsequent molecular dynamics simulations are labeled in blue.</p> <p>(D) Eigenvalue spectrum obtained from the principal component analysis of the experimentally determined conformers. The magnitude of each eigenvalue is expressed as the proportion of the total variance (mean-square fluctuation) captured by the corresponding eigenvector. Labels on each point indicate the cumulative sum of variance accounted for by a particular eigenvector and its preceding eigenvectors.</p></div

Cross-correlated motions of p53 DNA binding domain.

<div><p>(Upper panel) The anti-correlated motions (A-C) that are present in extended L1 but absent in recessed L1 conformers are colored in pink on the p53 DBD structures. The orientations of p53 DBD structures are the same as in Figure 1C. </p> <p>(Lower panel) The map of cross-correlated motions of extended L1 conformers (in upper triangle) and recessed L1 conformers (in lower triangle) shows different patterns between particular regions. For clarity, only correlated motions with absolute normalized values of at least 0.25 are shown. Motion occurring along the same direction is represented by positive correlation (cyan), whereas motion occurring along opposite directions is represented by negative (anti-) correlation (pink). </p></div

The sequences of BH3 peptide and stapled BH3 peptide analogs taken from Stewart [24] used in this study.

<p>The sequences of BH3 peptide and stapled BH3 peptide analogs taken from Stewart <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043985#pone.0043985-Stewart1" target="_blank">[24]</a> used in this study.</p

Binding free energy (in kcal/mol) of MCL-1 with wt and stapled BH3peptides.

<p>Binding free energy (in kcal/mol) of MCL-1 with wt and stapled BH3peptides.</p

BH3wt bound to MCL-1 (shown in grey).

<p>(A) Asp14 of the peptide interacts with Arg263 and Asn260 of MCL-1; Arg263 also interacts with Asp256; Arg10 hbonds with Ser255; His20 sidechain hbonds with the backbone of Phe318; Gln17 sidechain hbonds with the backbone of Gly262 (B) The hydrophobic groups Leu6, Leu9, and Val16 are buried in the hydrophobic binding groove on the surface of MCL-1 (shown in surface); BHC bound to MCL-1 (shown in grey) (C) The location of the staple forces it to point into the MCL-1 surface creating a steric clash, thus creating a strain on the backbone of the BH3C peptide and its helicity. The loss of key hbond networks result in decreased contributions from Arg10 and Asp14 when compared with BH3wt peptide (shown in cartoon), (D) MCL-1 bound to BH3C peptide (shown in surface); BH3H bound to MCL-1 (shown in grey) (E) Double stapling improves the packing of the stapled regions and also maintains the helical content (in cartoon) (F) The hydrophobic groups Leu6, Leu9, and Val16 are buried in the hydrophobic binding groove on the surface of MCL-1 (shown in surface); BH3K bound to MCL-1 (shown in grey) (G) This staple interacts with the hydrophobic patch on the MCL-1 surface but also enables Gln17 to stabilize the system by hbonding to the backbone of Gly262 (shown in cartoon) (H) The hydrophobic groups Leu6, Leu9, and Val16 are buried in the hydrophobic binding groove on the surface of MCL-1 (shown in surface).</p