Modelling the influence of ligand binding towards the structure and dynamics of protein arginine deiminase IV

An enzyme called Protein Arginine Deiminase IV (PAD4) has gained tremendous attention due to its role in rheumatoid arthritis (RA). It catalyses the citrullination reaction, whose products was reported to be dysregulated in the RA patients. Therefore, modulation of PAD4 activities is considered as an alternative therapeutic strategy against the disease. The early view of protein as a rigid body has been replaced by dynamics model. The use of dynamics model allows exploration of unique conformational changes that are varied depending on the protein environment. Although the structure of PAD4 has been solved experimentally, it does not reflect the dynamical changes of the protein. The present thesis employed molecular dynamics (MD) simulations to enhance understanding on PAD4 behaviour, and on how a ligand can influence the protein structure, dynamics and catalytic pocket. The MD simulations were performed in the ligand-bound and the unbound form of the enzyme. For ligand-bound systems, two known ligands were used: o-F-amidine and GSK147, which binds at two different binding regions. For the PAD4-GSK147 systems, the simulations were performed in the presence of five and two calcium ions conditions. This provided a platform for inspection and direct comparison of the structural and dynamics changes at the atomic level. The binding of either ligand showed a significant reduction in the local fluctuation profiles up to 30% at regions that are distal from the catalytic pocket, particularly residues in Subdomain I and 387-407, in comparison to that in the unbound-PAD4-5Ca2+ system. Comparison on the collective motion from the MD simulations revealed that the key of PAD4 inhibition by these ligands were through constraining the movement of Subdomain I in relative to Subdomain II, with two different hinge points. A reduction in the number of calcium ions in the PAD4-GSK147 simulation was also observed to greatly affect not only PAD4 structure and dynamics, but also protein-ligand interaction. This observation suggests that residues at the calcium binding sites can be utilised for modulating the enzyme. A clustering analysis on the PAD4 trajectories revealed the dynamics behaviour of H640 side chain, which is located in between the two door of PAD4 catalytic pocket. The upward movement of the H640 side chain changes the topography of the two-door catalytic pocket, which resembles an open-cleft event. This new insight on the H640 side chain has assisted in the identification of 47 drug-like compounds that exerts mean binding affinity in the range of -9.12 to -7.33 kcal/mol towards PAD4 and binds at either front door, back door or in between the two-door catalytic pocket. Binding interaction analyses of the top PAD4-binder complexes showed that the top binders were interacting to one or more key residues lining either the PAD4 front door or the back door; thus may be blocking the citrullination reaction. With additional optimisation, these binders may serve as lead compounds in future drug and development against PAD4. Overall, this work provides new and promising insights into the PAD4 dynamics and catalytic pocket.

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