Molecular Modeling Study for Interaction between Bacillus subtilis Obg and Nucleotides

The bacterial Obg proteins (Spo0B-associated GTP-binding protein) belong to the subfamily of P-loop GTPase proteins that contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain which is referred as the “Obg fold” and now it is considered as one of the new targets for antibacterial drug. When the Obg protein is associated with GTP, it becomes activated, because conformation of Obg fold changes due to the structural changes of GTPase switch elements in GTP binding site. In order to investigate the effects and structural changes in GTP bound to Obg and GTPase switch elements for activation, four different molecular dynamics (MD) simulations were performed with/without the three different nucleotides (GTP, GDP, and GDP + Pi) using the Bacillus subtilis Obg (BsObg) structure. The protein structures generated from the four different systems were compared using their representative structures. The pattern of Cα-Cα distance plot and angle between the two Obg fold domains of simulated apo form and each system (GTP, GDP, and GDP+Pi) were significantly different in the GTP-bound system from the others. The switch 2 element was significantly changed in GTP-bound system. Also root-mean-square fluctuation (RMSF) analysis revealed that the flexibility of the switch 2 element region was much higher than the others. This was caused by the characteristic binding mode of the nucleotides. When GTP was bound to Obg, its γ-phosphate oxygen was found to interact with the key residue (D212) of the switch 2 element, on the contrary there was no such interaction found in other systems. Based on the results, we were able to predict the possible binding conformation of the activated form of Obg with L13, which is essential for the assembly with ribosome

Structural origins for the loss of catalytic activities of bifunctional human LTA4H revealed through molecular dynamics simulations.

Human leukotriene A4 hydrolase (hLTA4H), which is the final and rate-limiting enzyme of arachidonic acid pathway, converts the unstable epoxide LTA4 to a proinflammatory lipid mediator LTB4 through its hydrolase function. The LTA4H is a bi-functional enzyme that also exhibits aminopeptidase activity with a preference over arginyl tripeptides. Various mutations including E271Q, R563A, and K565A have completely or partially abolished both the functions of this enzyme. The crystal structures with these mutations have not shown any structural changes to address the loss of functions. Molecular dynamics simulations of LTA4 and tripeptide complex structures with functional mutations were performed to investigate the structural and conformation changes that scripts the observed differences in catalytic functions. The observed protein-ligand hydrogen bonds and distances between the important catalytic components have correlated well with the experimental results. This study also confirms based on the structural observation that E271 is very important for both the functions as it holds the catalytic metal ion at its location for the catalysis and it also acts as N-terminal recognition residue during peptide binding. The comparison of binding modes of substrates revealed the structural changes explaining the importance of R563 and K565 residues and the required alignment of substrate at the active site. The results of this study provide valuable information to be utilized in designing potent hLTA4H inhibitors as anti-inflammatory agents

Overlay of WT and mutant systems of L-LTA4 complexes to observe the structural changes.

<p>(A) WT and E271Q systems, (B) WT and R563A systems, (C) WT and K565A systems, and (D) all L-LTA4 systems. The WT, E21Q, R563A, and K565A systems are shown in grey, wheat, deep teal, and pink colors, respectively. The amino acid residues and LTA4 are shown in think stick and ball-stick forms, respectively. The metal ion (zinc) present at the active site is shown in sphere form.</p

The initial binding poses.

<p>(A) LTA4 and (B) RAR selected from the molecular docking studies to be subjected to MD simulations.</p

Basic analyses to investigate the overall of behavior of the systems during the MD simulations.

<p>The RMSD, RMSF, and number of intramolecular hydrogen bonds are shown for (A) L-LTA4 (B) L-RAR systems.</p

The details of all systems subjected to MD simulations.

<p>The details of all systems subjected to MD simulations.</p

The 2D structures of two diverse substrates of bifunctional enzyme, LTA4H.

<p>The 2D structures of two diverse substrates of bifunctional enzyme, LTA4H.</p

The binding modes of RAR at the active site of the enzyme in L-RAR systems.

<p>The binding mode of RAR in (A) WT (cyan), (B) E271Q (light orange) (C) R563A (sand), and (D) K565A (blue white) systems. The amino acid residues and RAR are shown in think stick and ball-stick forms, respectively. The metal ion (zinc) present at the active site is shown in sphere form.</p

Overlay of WT and mutant systems of L-RAR complexes to observe the structural changes.

<p>(A) WT and E271Q systems, (B) WT and R563A systems, (C) WT and K565A systems, and (D) all L-RAR systems. The WT, E21Q, R563A, and K565A systems are shown in grey, wheat, deep teal, and pink colors, respectively. The amino acid residues and RAR are shown in think stick and ball-stick forms, respectively. The metal ion (zinc) present at the active site is shown in sphere form.</p

The binding modes of two substrates in the active site of the WT enzyme.

<p>(A) LTA4 and RAR substrates shown in grey and cyan at the active site (B) the catalytic active site residues are shown in thin stick form.</p