MUMBO energy score for total mutagenesis mapped to the 1YCR crystal structure.

<p>The p53 peptide is bound in the foreground. In red and blue ball-and-sticks, significantly energetically constrained and energetically mutable residues are shown, respectively, and are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147806#pone.0147806.t001" target="_blank">Table 1</a>. Residues shown in sticks are previously identified hot-spots determined through alanine-scanning mutagenesis (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147806#pone.0147806.t001" target="_blank">Table 1</a>) including the three directly interacting p53 residues. The backbone itself is colored by each position’s z-score, showing the relative constraint of each position along the backbone. The color scale is shown in the accompanying color scale bar, which ranges from energetically mutable (blue) to energetically constrained (red).</p

R65 to E69 Tract Average ΔΔG Compared to Average of Respective Wild Type ΔΔG.

<p>Average by position, dark grey bars. Average for respective wild type residue as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147806#pone.0147806.g003" target="_blank">Fig 3</a> with associated error bars, light grey.</p

Residues of Significance.

<p>Residues of Significance.</p

Dependence of prevalence of contiguous pathways in proteins on structural complexity

<div><p>Allostery is a regulatory mechanism in proteins where an effector molecule binds distal from an active site to modulate its activity. Allosteric signaling may occur via a continuous path of residues linking the active and allosteric sites, which has been suggested by large conformational changes evident in crystal structures. An alternate possibility is that the signal occurs in the realm of ensemble dynamics via an energy landscape change. While the latter was first proposed on theoretical grounds, increasing evidence suggests that such a control mechanism is plausible. A major difficulty for testing the two methods is the ability to definitively determine that a residue is directly involved in allosteric signal transduction. Statistical Coupling Analysis (SCA) is a method that has been successful at predicting pathways, and experimental tests involving mutagenesis or domain substitution provide the best available evidence of signaling pathways. However, ascertaining energetic pathways which need not be contiguous is far more difficult. To date, simple estimates of the statistical significance of a pathway in a protein remain to be established. The focus of this work is to estimate such benchmarks for the statistical significance of contiguous pathways for the null model of selecting residues at random. We found that when 20% of residues in proteins are randomly selected, contiguous pathways at the 6 Å cutoff level were found with success rates of 51% in PDZ, 30% in p53, and 3% in MutS. The results suggest that the significance of pathways may have system specific factors involved. Furthermore, the possible existence of false positives for contiguous pathways implies that signaling could be occurring via alternate routes including those consistent with the energetic landscape model.</p></div

Pathway success rates.

<p>Convergence of the successful formation of contiguous pathways was determined for each model system. The x-axis indicates the number of selections of random residues. The rate at which any pathway connecting the active and allosteric site, subject to the 6Å distance constraint, is shown on the y-axis. The horizontal line indicates the converged value. (A) PDZ (number of trials = 10 at each selection level), (B) p53 (number of trials = 3 at each selection level), (C) MutS (number of trials = 3 at each selection level).</p

PDZ contiguous pathway examples.

<p>The PDZ protein backbone is shown in a cyan tube (PDB ID 1BE9). The allosteric signal travels between the allosteric effector binding site to the peptide ligand binding site (residues Arg 313 (top) and Ala 376 (bottom) highlighted in red ball and stick representation). The 20% of residues randomly selected for the trial appear in dark blue ball and stick. In panels (A-D), lime green balls highlight residues forming a successful contiguous pathway at the 6Å cutoff level. A short, direct path of 4 (A) and 5 (B) residues connects the sites. Longer meandering paths were also discovered: 8 residues (C) and 14 residues (D). In panels (E) and (F), examples of failed pathways are shown. A red surface shows where the pathway failed to connect.</p

Success of pathways as a function of neighbor distance cutoff.

<p>The distance requirement for the contiguous pathway was varied (number of selections = 1000, number of trials = 3) for the three model systems, and was fit with a logistic curve. The best fit equations by system are given by system. PDZ: y = 100.663 + (5.015053–100.663)/(1 + (x/150.7424)^5.362259)^24925470 p53: y = 96.11989 + (0.4398672–96.11989)/(1 + (x/33.24754)^10.47436)^21720600 MutS: y = 100.1441 + (2.080954–100.1441)/(1 + (x/8.69924)^15.53203)^0.3183134.</p

Evolutionary Covariance Combined with Molecular Dynamics Predicts a Framework for Allostery in the MutS DNA Mismatch Repair Protein

Mismatch repair (MMR) is an essential, evolutionarily conserved pathway that maintains genome stability by correcting base-pairing errors in DNA. Here we examine the sequence and structure of MutS MMR protein to decipher the amino acid framework underlying its two key activitiesrecognizing mismatches in DNA and using ATP to initiate repair. Statistical coupling analysis (SCA) identified a network (sector) of coevolved amino acids in the MutS protein family. The potential functional significance of this SCA sector was assessed by performing molecular dynamics (MD) simulations for alanine mutants of the top 5% of 160 residues in the distribution, and control nonsector residues. The effects on three independent metrics were monitored: (i) MutS domain conformational dynamics, (ii) hydrogen bonding between MutS and DNA/ATP, and (iii) relative ATP binding free energy. Each measure revealed that sector residues contribute more substantively to MutS structure–function than nonsector residues. Notably, sector mutations disrupted MutS contacts with DNA and/or ATP from a distance via contiguous pathways and correlated motions, supporting the idea that SCA can identify amino acid networks underlying allosteric communication. The combined SCA/MD approach yielded novel, experimentally testable hypotheses for unknown roles of many residues distributed across MutS, including some implicated in Lynch cancer syndrome

WebLogo of Amino Acid Sequence of Circumsporozoite Protein from Malawi and the Gambia.

<p>Panel A and Panel B are the Weblogos for Malawi and the Gambia, respectively. In Panel A, the TH2 region (blue) and TH3 region (pink) are underlined. The TH2 epitope maps almost exclusively to the α-helix, while the TH3 epitope maps to the flap. The polymorphic residues and types of amino acids that populate these sites appear to be conserved between two geographically disparate African parasite populations. Bits represent the information content, which is a relative measurement of sequence conservation, with higher values representing conservation and lower values consistent with sequence diversity at a position.</p

Location of TH2 and TH3 epitopes in the Structure of Circumsporozoite Protein.

<p>The relative location of TH2 (blue) and TH3 (pink) are shown within the protein structure.</p