Normalized square fluctuation (nor. sq. flucs.) distribution.

<p>(<b>A</b>) Scatter plot of the normalized square fluctuation of all residues. The X-axis represents the normalized square fluctuation for MD and the Y-axis represents the normalized square fluctuation for ID. The solid line in the plot represents the unity line. Violin plots for (<b>B</b>) all residues (two-sample KS test, p-value < 2.2e-16), (<b>C</b>) interface residues (two-sample KS test, p-value < 2.2e-16) and, (D) functional residues (MD_f, ID_f, two-sample KS test, p-value: 5.4e-11) and non-functional residues (MD_nf, ID_nf, two-sample KS test, p-value < 2.2e-16).</p

Homologous domain pairs.

<p>Four homologous domain pairs namely (<b>A</b>) phosphoribosylanthranilate isomerase from <i>E</i>. <i>coli</i> and <i>Jonesia denitrificans</i>, (<b>B</b>) cyclophilin from <i>Bos taurus</i> and <i>Homo sapiens</i>, (<b>C</b>) sialidase from <i>Micromonospora viridifaciens</i> and <i>Homo sapiens</i> and, (<b>D</b>) hexokinase-1 from <i>Homo sapiens</i> and <i>Saccharomyces cerevisiae</i>. The top panel shows the multi-domain protein, the centre panel shows the single-domain protein and the last panel shows the superposition of homologous domains, with sequence identity (SI) and RMSD values.</p

Cross-correlation analysis of protein domain pairs.

<p>(<b>A</b>) Distribution of the cross-correlation values for 20 protein domain pairs. A higher number of residues show high positive cross-correlation values in MD as compared to ID. (<b>B</b>) Distribution of R_v coefficient for 20 protein domain pairs. Cartoon representation of few proteins along with the PDB id is shown above the corresponding R_v coefficient. The spheres represent the interface residues. (<b>C</b>) Cross-correlation matrices of fibronectin (FNIII 10 domain). The left panel is for MD and the right panel is for ID. The color bar represents the cross-correlation values.</p

Cross-correlation matrices of the functional residues.

<p><b>(A</b>) Phosphoribosylanthranilate isomerase, (<b>B</b>) cyclophilin, (<b>C</b>) sialidase and, (<b>D</b>) hexokinase-1. The first row represents the matrices for multi-domain proteins, the second row represents the matrices for single-domain and, the last row represents the chimeras. The X-axis and the Y-axis represent the residue number of the functional residues. The color bar represents the cross-correlation values.</p

Structural differences observed in the protein domain pairs.

<p>(<b>A</b>) RMSD and GDT distributions of the 20 protein domain pairs. The X-axis represents each domain pair in the dataset and the Y-axis represents the RMSD (left) and 100-GDT (right). (<b>B</b>) RMSD distribution of the protein domain pairs (colored cyan) and the control dataset 1 (colored pink). The distributions are significantly different, two-sample KS test; p-value: 1.26e-06. (<b>C</b>) 100-GDT distribution of the protein domain pairs (colored cyan) and the control dataset 1 (colored pink). The distributions are significantly different, two-sample KS test; p-value: 8.14e-06. (<b>D</b>) Distribution of the deviations observed for functional residues, interface residues and all the residues in the dataset. (<b>E</b>) Representative examples are shown for the types of local structural deviation observed in the dataset. The domain-domain interface regions are represented in sticks and the regions showing significant structural deviation are encircled in black.</p

Distribution of the normalized square fluctuation of the functional residues.

<p>Scatter plots for functional residues of (<b>A</b>) phosphoribosylanthranilate isomerase, (<b>B</b>) cyclophilin, (<b>C</b>) sialidase and, (<b>D</b>) hexokinase-1. The X-axis represents the normalized square fluctuation for multi-domain protein (MD) and the Y-axis represents the normalized square fluctuation for single-domain protein (SD). The solid line in all the plots represents the unity line.</p

Frustration index (FI) distribution.

<p>Scatter plot of the distribution of <b>(A)</b> single residue level frustration (SRLF) (two-sample KS test, p-value: 0.98), <b>(B)</b> mutational frustration index (MFI) (two-sample KS-test, p-value: 0.09) and, <b>(C)</b> configurational frustration index (CFI) (two-sample KS test, p-value: 0.63) of residues for all the domain pairs. The X-axis represents the frustration index for MD and the Y-axis represents the frustration index for ID. Residues which are minimally frustrated (MF) in both MD and ID are represented as green filled circles, residues which are neutrally frustrated (NF) in both MD and ID are represented as blue filled circles and residues which are highly frustrated (HF) in both MD and ID are represented as red filled circles. The residues showing differences in the type of frustration between MD and ID are represented as yellow filled circles. The dotted lines represent the cut-off used for the definition of frustration index (MF: FI ≥ 0.78; HF: FI ≤ -1 and NF: -1 < FI < 0.78). 3-D plot of the distribution of <b>(D)</b> frustration type, <b>(E)</b> fraction of highly mutationally frustrated contacts of each residue and, <b>(F)</b> fraction of highly configurationally frustrated contacts of each residue and the structural deviation observed between the corresponding C-alphas on superposition. A plane (grey color) is drawn at the deviation value of 1Å.</p

Normalized communicability centrality (coc) score distribution.

<p>Normalized coc distribution of <b>(A)</b> all the residues, (<b>B</b>) the interface residues and, (<b>C</b>) the non-interface residues of 20 protein domain pairs. The X-axis represents the normalized coc of MD and the Y-axis represents the normalized coc of ID. The solid line in the plots represents the unity line. (<b>D</b>) Normalized coc distribution of fibronectin (FNIII 10 domain). The red box encloses regions away from domain-domain interface region and the black boxes indicate the region around domain-domain interface. The X-axis represents the residue numbers and the Y-axis represents the normalized coc.</p

Design, Synthesis, and Experimental Validation of Peptide Ligands Targeting <i>Mycobacterium tuberculosis</i> σ Factors

Transcription in prokaryotes is a multistep process and is primarily regulated at the initiation stage. σ factors are involved in promoter recognition and thus govern prokaryotic gene expression. <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) σ factors have been previously suggested as important drug targets through large-scale genome analyses. Here we demonstrate the feasibility of specific targeting of <i>Mtb</i> σ factors using designed peptides. A peptide library was generated using three-dimensional structural features corresponding to the interface regions of σ factors and the RNA polymerase. <i>In silico</i> optimization of the peptides, employing structural as well as sequence features, aided specific targeting of σ^A and σ^B. We synthesized and characterized the best hit peptide from the peptide library along with other control peptides and studied the interaction of these peptides with σ^B using biolayer interferometry. The experimental data validate the design strategy. These studies suggest the feasibility of designing specific peptides via <i>in silico</i> methods that bind σ^B with nanomolar affinity. We note that this strategy can be broadly applied to modulate prokaryotic transcription by designed peptides, thereby providing a tool for studying bacterial adaptation as well as host–pathogen interactions in infectious bacteria