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Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

Shi J, Han N, Lim L, Lua S, Sivaraman J, Wang L, Mu Y, Song J - PLoS Comput. Biol. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.

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.

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Related in: MedlinePlus

Enzymatic activity and dissociation constant of the dimer-monomer equilibrium.(a). Enzymatic activities of the WT (black lines) and N214A (dotted lines) proteases by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm continuously for 3 min. The Km and kcat values are presented for the WT enzyme. The ITC dilution profiles for measuring the dissociation constants of the dimer-monomer equilibrium for WT (b) and N214A (c). The Kd and ΔH values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software.
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pcbi-1001084-g001: Enzymatic activity and dissociation constant of the dimer-monomer equilibrium.(a). Enzymatic activities of the WT (black lines) and N214A (dotted lines) proteases by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm continuously for 3 min. The Km and kcat values are presented for the WT enzyme. The ITC dilution profiles for measuring the dissociation constants of the dimer-monomer equilibrium for WT (b) and N214A (c). The Kd and ΔH values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software.

Mentions: By a fluorescence resonance energy transfer (FRET)-based assay, we have measured the enzymatic activities of both WT and N214A proteases. As shown in Figure 1a, the WT protease is fully active with the Km and kcat values very similar to that previously reported on the authentic enzyme [17]. However, the activity of the N214A mutant is extremely low and consequently had no detectable increase of the fluorescence intensity within 3-minute incubation. Only after 2 hours, a slight increase of the fluorescence intensity could be detected (data not shown). We have also tested on the activity at higher N214A concentrations, no significant activity enhancement was observed. Because of this, we were unable to fit out their precise Km and kcat values although we collected a large set of data for N214A.


Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

Shi J, Han N, Lim L, Lua S, Sivaraman J, Wang L, Mu Y, Song J - PLoS Comput. Biol. (2011)

Enzymatic activity and dissociation constant of the dimer-monomer equilibrium.(a). Enzymatic activities of the WT (black lines) and N214A (dotted lines) proteases by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm continuously for 3 min. The Km and kcat values are presented for the WT enzyme. The ITC dilution profiles for measuring the dissociation constants of the dimer-monomer equilibrium for WT (b) and N214A (c). The Kd and ΔH values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044768&req=5

pcbi-1001084-g001: Enzymatic activity and dissociation constant of the dimer-monomer equilibrium.(a). Enzymatic activities of the WT (black lines) and N214A (dotted lines) proteases by monitoring the increase of the emission fluorescence intensity at a wavelength of 538 nm continuously for 3 min. The Km and kcat values are presented for the WT enzyme. The ITC dilution profiles for measuring the dissociation constants of the dimer-monomer equilibrium for WT (b) and N214A (c). The Kd and ΔH values were obtained by fitting the ITC data with the built-in Microcal ORIGIN software.
Mentions: By a fluorescence resonance energy transfer (FRET)-based assay, we have measured the enzymatic activities of both WT and N214A proteases. As shown in Figure 1a, the WT protease is fully active with the Km and kcat values very similar to that previously reported on the authentic enzyme [17]. However, the activity of the N214A mutant is extremely low and consequently had no detectable increase of the fluorescence intensity within 3-minute incubation. Only after 2 hours, a slight increase of the fluorescence intensity could be detected (data not shown). We have also tested on the activity at higher N214A concentrations, no significant activity enhancement was observed. Because of this, we were unable to fit out their precise Km and kcat values although we collected a large set of data for N214A.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore.

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.

Show MeSH
Related in: MedlinePlus