Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Abstract

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.

Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations

“…It is well established that helices can be stabilized by local interactions while β-sheets are mostly specified by long-range interactions and their presence is, therefore, highly context-dependent. Consequently, loss of long-range interactions usually leads to the chameleon transformation into helical conformations as exemplified by our previous reports [29], [65], [66].…”

“…To unravel the intrinsic dynamics of the EphA5 LBD, three independent, 30 ns molecular dynamics simulations were performed as we previously conducted [14], [25], [29]. Briefly, the simulation cell is a periodic cubic box with a minimum distance of 9 Å between the protein and the box walls to ensure the proteins does not directly interact with its own periodic image.…”

Structural, Stability, Dynamic and Binding Properties of the ALS-Causing T46I Mutant of the hVAPB MSP Domain as Revealed by NMR and MD Simulations

“…This implies that the protein dynamics may play a key role beyond the static structure, as we recently demonstrated on the SARS 3C-like protease [36]. As such here we utilized the molecular dynamics (MD) simulation to explore the dynamical behavior of the MSP domain as well as the consequence of the T46I mutation.…”

“…The T46I mutation on the MSP domain was generated by use of site-directed mutagenesis [36]. The vectors were transformed into E. coli BL21 (DE3) cells (Novagen) for protein expression.…”

Unique properties of Zika NS2B-NS3pro complexes as decoded by experiments and MD simulations

“…Molecular dynamics (MD) simulation represents a powerful tool to gain insights into roles of protein dynamics in the enzymatic catalysis (28), and we have previously utilized it to study the Dengue NS2B-NS3pro in the "open" form (12), as well as the dynamically-driven allosteric mechanisms of the SARS 3C-like protease, which also shares the chymotrypsin fold to host the catalytic machinery (29,30). Here with the exact same protocols we previously used for the Dengue (12), we conducted MD simulations up to 100 ns for Zika and Dengue NS2B-author/funder.…”