Table 1.
Experimentally verified binding motifs (indicated by underline) of DYNLL1 interacting partners.
Figure 1.
Schematic illustration of strategy.
(1) Collected DYNLL1 binding motif sequences and PDBSum database was screened for these motifs. (2) Candidate proteins containing the DYNLL1 interaction motifs were picked (Table 2, bold hits). The selected proteins were tested by molecular docking against DYNLL1 to verify the hits which bind to DYNLL1 specific binding pocket. (3) Selected DYNLL1-interacting proteins on the basis of binding score values and common docking poses. (4) 3D structure comparison of DYNLL1 binding partners was carried out to examine the motif similarity pattern. (5) 3D structure comparison among known and novel DYNLL1 interacting proteins was conducted to inspect the structural conservation of putative DYNLL1 binding motifs and to evaluate the similarity of interacting mode. (6) Candidate proteins exhibiting highly conserved and identical binding pattern to the known one were selected. (7) Finally, annotated the possible pathways for the functional basis of novel interactions.
Table 2.
DYNLL1 binding motif containing viral proteins.
Figure 2.
Conservation pattern of Pilin receptor binding domain.
Pilin receptor binding domains (128-144 AA) of different strains of P. aeruginosa [60] are indicated and aligned. The Pilin protein of PAO strain contains a putative DYNLL1 binding motif which is highlighted by red rectangle. The black and dark gray colors show motif conservation pattern among four strains of P. aeruginosa, respectively.
Figure 3.
Residual contributions of DYNLL1 and Pilin explored through multiple docking protocols.
The interacting residues of (A) DYNLL1 binding groove and (B) Pilin receptor binding domain are indicated. The residues marked in blue color are common among all the best docking poses, yellow and green colors indicate the residues which are common in 3 and 4 poses, respectively out of the 5 docking results. Pilin specific motif (KSTQD) is shown by black rectangle. Red rectangle highlights Lys130 residue which is only present in PAO strain. The docking/binding energy values for DYNLL1-Pilin obtained through individual docking procedures are: AUTODOCK, 0.9 kcal/mol; PatchDock, −28.31 kcal/mol; ZDOCK, −20.76 kcal/mol; DOCK/PIERR, −37.08 kcal/mol; CLUSPRO, −647.5 kcal/mol, respectively.
Figure 4.
Visualization of the best docked DYNLL1-Pilin complex in dimer form.
Blue and orange ribbons indicate individual DYNLL1 monomers connected by H-bonds as indicated. Pilin is shown in ribbon form in pink and yellow colors. Interacting residues are shown in atomic representations and H-bonds are shown by dotted black lines. Pilin specific residues (Lys130, Thr132 and Asp134) are involved in hydrogen bonding.
Figure 5.
Structural comparisons of all possible combinations of binding motifs.
Individual RMSD values are plotted against the corresponding superposed motifs to all the indicated proteins including Pilin (KSTQD), Adenain (KSTQT), BimEL (KSTQT), DNMT3A (LGIQV), EML3 (RGTQT), nNOS (TGIQV), PAK1 (DVATS), Rack1 (YTVQD), P protein Mokola virus (KSTQT), P protein Rabies virus (KSTQT) and Vaccinia polymerase (KQTQT). The RMSD analysis shows that Pilin specific motif contains a close structural similarity to the validated DYNLL1 binding motifs.
Figure 6.
Stability and residue fluctuations of 15(DYNLL1-Pilin).
(A) RMSD plot of Cα atoms computed through bound DYNLL1-Pilin complex (blue) and unbound structure (red). (B) Comparison of the bound (blue) and unbound (red) structures quantified by their backbone root-mean-square-fluctuations (RMSF). The pilin binding site is indicated. (C) Comparative RMSF plot for DYNLL1-loop region between Arg60 and Gln80. (D) Radius of gyration (Rg) plot for DYNLL1-Pilin complex.
Figure 7.
Structural and conformational adjustments of DYNLL1-Pilin system at different time scales.
Snapshots were collected throughout the MD simulations of each system and PDBs were generated for 1, 5, 10, 12 and 15-dependent behavior and stability of each system. PDB structures were visualized in UCSF Chimera 1.7.0. 2D plots were generated through DIMPLOT and residues involved hydrogen bonding (indicated by black dotted lines) were monitored. The Pilin specific residues involved in hydrogen bonding are indicated by joining pink dotted circles. The observed DYNLL1-loop measurements were 24.62 Å, 24.49 Å, 27.83 Å, 25.07 Å and 23.36 Å, respectively. The binding energy values at indicated time scales were −5384.58 kJ/mol, −5100.22 kJ/mol, −4848.70 kJ/mol, −5017.51 kJ/mol and 5003.91 kJ/mol, respectively. Superimposed structures at the indicated time intervals for DYNLL1 are indicated by pink (1 ns), cyan (5 ns), blue (10 ns), pink (12 ns) and yellow (15 ns) colors, respectively.
Figure 8.
Roles of DYNLL1 and Pilin in host defense mechanism.
Model of normal cell host defense mechanism. Pilin and flagellin trigger inflammasome activity in NLRC4 inflammasome activation pathway. DYNLL1/LC8 regulates NF-κβ activation by inhibiting the phosphorylation of Iκβα by Iκβ kinase.
Figure 9.
Role of DYNLL1-Pilin interaction during chronic inflammation.
Model of P. aeruginosa infected cell pathway. (1) DYNLL1/LC8-Pilin interaction interrupts the reduction mechanism of TRP14 by maintaining DYNLL1 in oxidized homodimeric form which is unable to bind with Iκβα. Iκβα is instantly degraded due to continuous phosphorylation by Iκβ kinase, resulting in up regulation of NF-κβ pathway. (2) Pilin hijacks DYNLL1 to obtain compartmental specificity and inflammasome activation.