Molecular interaction of ATP with zNOD1 (A), zNOD2 (B) and mNLRC4-NACHT after molecular dynamics simulation.

<p>The critical amino acids involved in ATP binding are highlighted in bold font.</p

Variation of H-bonds participated in interaction during 50 ns simulation in zNOD-NACHT complexes.

<p>(A) Complex I for zNOD1-NACHT, (B) Complex II for zNOD1-NACHT, (C) Complex III for zNOD1-NACHT, (D) Complex I for zNOD2-NACHT, (E) Complex II for zNOD2-NACHT and (F) Complex III for zNOD2-NACHT (G) mNLRC4-NACHT complex. The y-axis represents the number of H-bond formed during the course of MD simulation and the simulation time (in ns) is depicted in x-axis. All graphs are generates using Grace 5.1.23 plotting program.</p

Interaction analysis (hydrogen bond, hydrophobic contacts, electrostatics and van der Waals contact) of NOD1-NACHT-ATP and NOD2-NACHT-ATP complexes before molecular dynamics simulation.

<p>Interaction analysis (hydrogen bond, hydrophobic contacts, electrostatics and van der Waals contact) of NOD1-NACHT-ATP and NOD2-NACHT-ATP complexes before molecular dynamics simulation.</p

Molecular interaction of ATP with zNOD1 and zNOD2-NACHT domains.

<p>(A) Complex I for NOD1-NACHT, (B) Complex II for NOD1-NACHT, (C) Complex III for NOD1-NACHT, (D) Complex I for NOD2-NACHT, (E) Complex II for NOD1-NACHT, and (F) Complex III for NOD1-NACHT. The graphics is generated using PyMOL. The ATP molecule is shown as stick, protein as cartoon, and H-bonds as red dotted lines. The amino acids marked in green are designated as H-bond forming residues.</p

Domain architectures of zNOD1 (A) and zNOD2 (B).

<p>The NACHT domain is characterized by five different functional motifs. Multiple sequence alignment of zebrafish, human and mouse NACHT domains is constructed using MAFFT and the conserved functional motifs are highlighted. The highlighted residues represent the potential ATP binding sites and the conserved residues are shown as ‘*’. The ATP-binding residues conserved in all three species are labeled.</p

ATP binding poses in zNOD-NACHT domains in simulated models zNOD1-NACHT-ATP complex (A), zNOD2-NACHT-ATP complex (B) and mNLRC4-NACHT-ATP complex (C).

<p>The protein is shown as cartoon; interacting amino acids are shown as lines and ATP as stick.9D) Multiple sequence alignment of NACHT domains of NOD1 and NOD2 (from human, mouse and zebrafish)with mouse NLRC4 sequences. The key functional motifs are underlined and labeled. The potential ATP binding residues are pointed by different colored triangles.</p

Conformational stability of NACHT-ATP complex throughout 50 ns time period.

<p>(A) Backbone-RMSD of zNOD1-NACHT, (B) Backbone-RMSD of zNOD2-NACHT, (C) Backbone-RMSD of mNLRC4-NACHT, (D) RMSD of ATP atoms in zNOD1-NACHT complex, (E) RMSD of ATP atoms in zNOD2-NACHT complex, (F) RMSD of ATP atoms in mNLRC4-NACHT complex (G) Radius of gyration (Rg) of zNOD1-NACHT, (H) Radius of gyration (Rg) of zNOD2-NACHT,and (F) Rg of mNLRC4-NACHT. All graphs are generated using Grace 5.1.23 plotting program.</p

Structural Models of Zebrafish (<i>Danio rerio</i>) NOD1 and NOD2 NACHT Domains Suggest Differential ATP Binding Orientations: Insights from Computational Modeling, Docking and Molecular Dynamics Simulations

<div><p>Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and NOD2 are cytosolic pattern recognition receptors playing pivotal roles in innate immune signaling. NOD1 and NOD2 recognize bacterial peptidoglycan derivatives iE-DAP and MDP, respectively and undergoes conformational alternation and ATP-dependent self-oligomerization of NACHT domain followed by downstream signaling. Lack of structural adequacy of NACHT domain confines our understanding about the NOD-mediated signaling mechanism. Here, we predicted the structure of NACHT domain of both NOD1 and NOD2 from model organism zebrafish (<i>Danio rerio</i>) using computational methods. Our study highlighted the differential ATP binding modes in NOD1 and NOD2. In NOD1, γ-phosphate of ATP faced toward the central nucleotide binding cavity like NLRC4, whereas in NOD2 the cavity was occupied by adenine moiety. The conserved ‘Lysine’ at Walker A formed hydrogen bonds (H-bonds) and Aspartic acid (Walker B) formed electrostatic interaction with ATP. At Sensor 1, Arg328 of NOD1 exhibited an H-bond with ATP, whereas corresponding Arg404 of NOD2 did not. ‘Proline’ of GxP motif (Pro386 of NOD1 and Pro464 of NOD2) interacted with adenine moiety and His511 at Sensor 2 of NOD1 interacted with γ-phosphate group of ATP. In contrast, His579 of NOD2 interacted with the adenine moiety having a relatively inverted orientation. Our findings are well supplemented with the molecular interaction of ATP with NLRC4, and consistent with mutagenesis data reported for human, which indicates evolutionary shared NOD signaling mechanism. Together, this study provides novel insights into ATP binding mechanism, and highlights the differential ATP binding modes in zebrafish NOD1 and NOD2.</p></div

The 3D models of zNOD1-NACHT (A) and zNOD2-NACHT (B) domain.

<p>The protein is shown as solid ribbon with α-helices (in red), β-sheets (in cyan), and turns (in white). The five different functional motifs are highlighted with different colors and are labeled. Walker A is shown in blue, Walker B in magenta, Sensor 1 in green, GxP motif in deep green, and Sensor 2 in deep blue. The ATP binding pocket is displayed with dashed line.</p

The figure indicates intermolecular interaction and distance analysis of ATP with the final representative structure of mNLRC4-NACHT obtained after 50 ns MD simulation.

<p>The distance of each observed H-bonds, specific electrostatic interaction and minimum contact between ATP and residue. The colors in boxes indicate different interactions where red indicates H-bond interaction, violet designates electrostatic interaction, and magenta represents close contacts.</p