Severe acute respiratory syndrome (SARS) coronavirus
is a novel human coronavirus and is responsible for
SARS infection. SARS coronavirus 3C-like proteinase
(SARS 3CLpro) plays key roles in viral replication and
transcription and is an attractive target for anti-SARS
drug discovery. In this report, we quantitatively characterized
the dimerization features of the full-length
and N-terminal residues 17 deleted SARS 3CLpros by
using glutaraldehyde cross-linking SDS-PAGE, size-exclusion
chromatography, and isothermal titration calorimeter
techniques. Glutaraldehyde cross-linking SDSPAGE
and size-exclusion chromatography results show
that, similar to the full-length SARS 3CLpro, the N-terminal
deleted SARS 3CLpro still remains a dimer/monomer
mixture within a wide range of protein concentrations.
Isothermal titration calorimeter determinations
indicate that the equilibrium dissociation constant (Kd)
of the N-terminal deleted proteinase dimer (262 M) is
very similar to that of the full-length proteinase dimer
(227 M). Enzymatic activity assay using the fluorescence
resonance energy transfer method reveals that
N-terminal deletion results in almost complete loss of
enzymatic activity for SARS 3CLpro. Molecular dynamics
and docking simulations demonstrate the N-terminal
deleted proteinase dimer adopts a state different from
that of the full-length proteinase dimer, which increases
the angle between the two protomers and reduces the
binding pocket that is not beneficial to the substrate
binding. This conclusion is verified by the surface plasmon
resonance biosensor determination, indicating
that the model substrate cannot bind to the N-terminal
deleted proteinase. These results suggest the N terminus
is not indispensable for the proteinase dimerization but
may fix the dimer at the active state and is therefore
vital to enzymatic activity. 等温滴定微量热仪(生物大分子相互作用仪) ITC ,MicroCal
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