The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain.
Identifieur interne : 001379 ( Ncbi/Checkpoint ); précédent : 001378; suivant : 001380The catalysis of the SARS 3C-like protease is under extensive regulation by its extra domain.
Auteurs : Jiahai Shi [Singapour] ; Jianxing SongSource :
- The FEBS journal [ 1742-464X ] ; 2006.
Descripteurs français
- KwdFr :
- Catalyse, Cysteine endopeptidases (), Cysteine endopeptidases (génétique), Cysteine endopeptidases (métabolisme), Dichroïsme circulaire, Diffusion de rayonnements, Dimérisation, Domaine catalytique, Lumière, Modèles moléculaires, Mutagenèse dirigée, Protéines de fusion recombinantes (), Protéines de fusion recombinantes (génétique), Protéines de fusion recombinantes (métabolisme), Protéines virales (), Protéines virales (génétique), Protéines virales (métabolisme), Résonance magnétique nucléaire biomoléculaire, Structure quaternaire des protéines, Structure tertiaire des protéines, Virus du SRAS (enzymologie), Virus du SRAS (génétique).
- MESH :
- enzymologie : Virus du SRAS.
- génétique : Cysteine endopeptidases, Protéines de fusion recombinantes, Protéines virales, Virus du SRAS.
- métabolisme : Cysteine endopeptidases, Protéines de fusion recombinantes, Protéines virales.
- Catalyse, Cysteine endopeptidases, Dichroïsme circulaire, Diffusion de rayonnements, Dimérisation, Domaine catalytique, Lumière, Modèles moléculaires, Mutagenèse dirigée, Protéines de fusion recombinantes, Protéines virales, Résonance magnétique nucléaire biomoléculaire, Structure quaternaire des protéines, Structure tertiaire des protéines.
English descriptors
- KwdEn :
- Catalysis, Catalytic Domain, Circular Dichroism, Cysteine Endopeptidases (chemistry), Cysteine Endopeptidases (genetics), Cysteine Endopeptidases (metabolism), Dimerization, Light, Models, Molecular, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Quaternary, Protein Structure, Tertiary, Recombinant Fusion Proteins (chemistry), Recombinant Fusion Proteins (genetics), Recombinant Fusion Proteins (metabolism), SARS Virus (enzymology), SARS Virus (genetics), Scattering, Radiation, Viral Proteins (chemistry), Viral Proteins (genetics), Viral Proteins (metabolism).
- MESH :
- chemical , chemistry : Cysteine Endopeptidases, Recombinant Fusion Proteins, Viral Proteins.
- chemical , genetics : Cysteine Endopeptidases, Recombinant Fusion Proteins, Viral Proteins.
- chemical , metabolism : Cysteine Endopeptidases, Recombinant Fusion Proteins, Viral Proteins.
- enzymology : SARS Virus.
- genetics : SARS Virus.
- Catalysis, Catalytic Domain, Circular Dichroism, Dimerization, Light, Models, Molecular, Mutagenesis, Site-Directed, Nuclear Magnetic Resonance, Biomolecular, Protein Structure, Quaternary, Protein Structure, Tertiary, Scattering, Radiation.
Abstract
The 3C-like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C-terminal extra domain in addition to the chymotrypsin-fold adopted by picornavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra-domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained approximately 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284-Thr285-Ile286 and another around Phe291) were discovered on which Ala-mutations significantly increased the enzymatic activities. Based on this, a super-active triple-mutant STI/A with a 3.7-fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild-type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization-critical residues plus Ser284-Thr285-Ile286 and Phe291 are clustered together to form a nano-scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.
DOI: 10.1111/j.1742-4658.2006.05130.x
PubMed: 16478476
Affiliations:
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pubmed:16478476Le document en format XML
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<term>Catalytic Domain</term>
<term>Circular Dichroism</term>
<term>Cysteine Endopeptidases (chemistry)</term>
<term>Cysteine Endopeptidases (genetics)</term>
<term>Cysteine Endopeptidases (metabolism)</term>
<term>Dimerization</term>
<term>Light</term>
<term>Models, Molecular</term>
<term>Mutagenesis, Site-Directed</term>
<term>Nuclear Magnetic Resonance, Biomolecular</term>
<term>Protein Structure, Quaternary</term>
<term>Protein Structure, Tertiary</term>
<term>Recombinant Fusion Proteins (chemistry)</term>
<term>Recombinant Fusion Proteins (genetics)</term>
<term>Recombinant Fusion Proteins (metabolism)</term>
<term>SARS Virus (enzymology)</term>
<term>SARS Virus (genetics)</term>
<term>Scattering, Radiation</term>
<term>Viral Proteins (chemistry)</term>
<term>Viral Proteins (genetics)</term>
<term>Viral Proteins (metabolism)</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Catalyse</term>
<term>Cysteine endopeptidases ()</term>
<term>Cysteine endopeptidases (génétique)</term>
<term>Cysteine endopeptidases (métabolisme)</term>
<term>Dichroïsme circulaire</term>
<term>Diffusion de rayonnements</term>
<term>Dimérisation</term>
<term>Domaine catalytique</term>
<term>Lumière</term>
<term>Modèles moléculaires</term>
<term>Mutagenèse dirigée</term>
<term>Protéines de fusion recombinantes ()</term>
<term>Protéines de fusion recombinantes (génétique)</term>
<term>Protéines de fusion recombinantes (métabolisme)</term>
<term>Protéines virales ()</term>
<term>Protéines virales (génétique)</term>
<term>Protéines virales (métabolisme)</term>
<term>Résonance magnétique nucléaire biomoléculaire</term>
<term>Structure quaternaire des protéines</term>
<term>Structure tertiaire des protéines</term>
<term>Virus du SRAS (enzymologie)</term>
<term>Virus du SRAS (génétique)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Recombinant Fusion Proteins</term>
<term>Viral Proteins</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Recombinant Fusion Proteins</term>
<term>Viral Proteins</term>
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<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cysteine Endopeptidases</term>
<term>Recombinant Fusion Proteins</term>
<term>Viral Proteins</term>
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<keywords scheme="MESH" qualifier="enzymologie" xml:lang="fr"><term>Virus du SRAS</term>
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<keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>SARS Virus</term>
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<keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>SARS Virus</term>
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<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cysteine endopeptidases</term>
<term>Protéines de fusion recombinantes</term>
<term>Protéines virales</term>
<term>Virus du SRAS</term>
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<term>Protéines de fusion recombinantes</term>
<term>Protéines virales</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Catalysis</term>
<term>Catalytic Domain</term>
<term>Circular Dichroism</term>
<term>Dimerization</term>
<term>Light</term>
<term>Models, Molecular</term>
<term>Mutagenesis, Site-Directed</term>
<term>Nuclear Magnetic Resonance, Biomolecular</term>
<term>Protein Structure, Quaternary</term>
<term>Protein Structure, Tertiary</term>
<term>Scattering, Radiation</term>
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<term>Cysteine endopeptidases</term>
<term>Dichroïsme circulaire</term>
<term>Diffusion de rayonnements</term>
<term>Dimérisation</term>
<term>Domaine catalytique</term>
<term>Lumière</term>
<term>Modèles moléculaires</term>
<term>Mutagenèse dirigée</term>
<term>Protéines de fusion recombinantes</term>
<term>Protéines virales</term>
<term>Résonance magnétique nucléaire biomoléculaire</term>
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<front><div type="abstract" xml:lang="en">The 3C-like protease of the severe acute respiratory syndrome (SARS) coronavirus has a C-terminal extra domain in addition to the chymotrypsin-fold adopted by picornavirus 3C proteases hosting the complete catalytic machinery. Previously we identified the extra domain to be involved in enzyme dimerization which has been considered essential for the catalytic activity. In an initial attempt to map out the extra-domain residues critical for dimerization, we have systematically generated 15 point mutations, five deletions and one triple mutation and subsequently characterized them by enzymatic assay, dynamic light scattering, CD and NMR spectroscopy. The results led to identification of four regions critical for enzyme dimerization. Interestingly, Asn214Ala mutant with a significant tendency to form a monomer still retained approximately 30% activity, indicating that the relationship between the activity and dimerization might be very complex. Very surprisingly, two regions (one over Ser284-Thr285-Ile286 and another around Phe291) were discovered on which Ala-mutations significantly increased the enzymatic activities. Based on this, a super-active triple-mutant STI/A with a 3.7-fold activity enhancement was thus engineered by mutating residues Ser284, Thr285 and Ile286 to Ala. The dynamic light scattering, CD and NMR characterizations indicate that the wild-type (WT) and STI/A mutant share similar structural and dimerization properties, thus implying that in addition to dimerization, the extra domain might have other mechanisms to regulate the catalytic machinery. We rationalized these results based on the enzyme structure and consequently observed an interesting picture: the majority of the dimerization-critical residues plus Ser284-Thr285-Ile286 and Phe291 are clustered together to form a nano-scale channel passing through the central region of the enzyme. We therefore speculate that this channel might play a role in relaying regulatory effects from the extra domain to the catalytic machinery.</div>
</front>
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