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Solution structure of the 2A protease from a common cold agent, human rhinovirus C2, strain W12.

Lee W, Watters KE, Troupis AT, Reinen NM, Suchy FP, Moyer KL, Frederick RO, Tonelli M, Aceti DJ, Palmenberg AC, Markley JL - PLoS ONE (2014)

Bottom Line: The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues.The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%.Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

View Article: PubMed Central - PubMed

Affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

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SDS-PAGE illustrating purification of RV-C2 2Apro.The recombinant methods described above were used to prepare 13C/15N-labeled C2 2Apro (C105A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH4)2SO4 precipitation; lane 3, SUMO-2Apro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.
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pone-0097198-g002: SDS-PAGE illustrating purification of RV-C2 2Apro.The recombinant methods described above were used to prepare 13C/15N-labeled C2 2Apro (C105A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH4)2SO4 precipitation; lane 3, SUMO-2Apro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.

Mentions: Cell pastes (5–10 g) were thawed and resuspended in lysis buffer (60–70 mL, 20 mM Tris pH 7.2, 500 mM NaCl, 10% ethylene glycol, 5 mM imidazole, 1 mM PMSF, 0.1% NP-40, Sigma) containing lysozyme (5 μL, Novagen), RNase (10 μL, Qiagen), Benzonase (5 μL, Novagen, 25 U/µl), or OmniCleave nuclease (Epicenter, 10 KU). The lysates were sonicated in a Misonix 3000 at 4°C with pulsing on (∼80 Watt) for 2 s and off for 4 s over 15 min and then clarified by centrifugation (30 min, 70,000 g). Polyethylene imine (to 0.1% w/v, Fluka) was added, and the samples were clarified again by centrifugation (30 min, 70,000 g) before the addition of (NH4)2SO4 (to 70% w/v) and DTT (to 2 mM). The collected pellets were resuspended in IMAC buffer 1 (30–40 mL, 20 mM Tris, pH 7.2, 10% glycerol, 35 mM imidazole, 1 mM PMSF), clarified (70,000 g, 30 min) then filtered (0.8 micron, Millipore) before loading onto IMAC resin (Qiagen Superflow FF) at a rate of 1–2 mL/min. The column (∼10 mL) was washed (10 volumes) with IMAC buffer 2 (buffer 1 plus 500 mM NaCl) then with IMAC buffer 3 (buffer 2 plus 65 mM imidazole), before protein elution with IMAC buffer 4 (buffer 2 plus 250 mM imidazole). Usually, 90% of the target was eluted in the first 15–30 mL as assayed by SDS-PAGE. Appropriate fractions were dialyzed overnight into buffer (Tris 20 mM pH 8.0, 150 mM NaCl and 2 mM DTT or β-mercaptoethanol), before the SUMO domain was removed from the N-terminus of 2Apro by incubation with 0.5 mg SUMO protease (prepared in house) for 3–4 h at 30°C. The sample was loaded onto an IMAC column freshly equilibrated with IMAC buffer 1, which bound the His-tagged SUMO domain. The 2Apro target was retrieved in the flow-through (4–5 fractions of 5–10 mL) and pooled. The final fractionation was by gel filtration (GE Healthcare HiPrep 16/60 Sephacryl S-200, 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM DTT). The purified protein was spin concentrated (Sartorius Vivaspin 20 10 kDa PES concentrator, 5,000 g) and then drop frozen in liquid nitrogen. The final yield was 27.5 mg of purified protein from 0.5 L double-labeled Martek (rich) media. The purity of protein samples was determined by SDS-PAGE (Figure 2). The C105A variant protein aggregated less during purification and produced a higher yield of protein.


Solution structure of the 2A protease from a common cold agent, human rhinovirus C2, strain W12.

Lee W, Watters KE, Troupis AT, Reinen NM, Suchy FP, Moyer KL, Frederick RO, Tonelli M, Aceti DJ, Palmenberg AC, Markley JL - PLoS ONE (2014)

SDS-PAGE illustrating purification of RV-C2 2Apro.The recombinant methods described above were used to prepare 13C/15N-labeled C2 2Apro (C105A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH4)2SO4 precipitation; lane 3, SUMO-2Apro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061012&req=5

pone-0097198-g002: SDS-PAGE illustrating purification of RV-C2 2Apro.The recombinant methods described above were used to prepare 13C/15N-labeled C2 2Apro (C105A) for NMR studies. Representative samples from the procedure were fractionated by SDS-PAGE then visualized with Bio-Rad Stain-Free. Lane 1, Bio-Rad Precision Plus protein standards; lane 2, protein pellet after (NH4)2SO4 precipitation; lane 3, SUMO-2Apro after IMAC elution; lane 4, 2Apro after SUMO cleavage and IMAC elution; lanes 5–6, final protein fractions after gel filtration.
Mentions: Cell pastes (5–10 g) were thawed and resuspended in lysis buffer (60–70 mL, 20 mM Tris pH 7.2, 500 mM NaCl, 10% ethylene glycol, 5 mM imidazole, 1 mM PMSF, 0.1% NP-40, Sigma) containing lysozyme (5 μL, Novagen), RNase (10 μL, Qiagen), Benzonase (5 μL, Novagen, 25 U/µl), or OmniCleave nuclease (Epicenter, 10 KU). The lysates were sonicated in a Misonix 3000 at 4°C with pulsing on (∼80 Watt) for 2 s and off for 4 s over 15 min and then clarified by centrifugation (30 min, 70,000 g). Polyethylene imine (to 0.1% w/v, Fluka) was added, and the samples were clarified again by centrifugation (30 min, 70,000 g) before the addition of (NH4)2SO4 (to 70% w/v) and DTT (to 2 mM). The collected pellets were resuspended in IMAC buffer 1 (30–40 mL, 20 mM Tris, pH 7.2, 10% glycerol, 35 mM imidazole, 1 mM PMSF), clarified (70,000 g, 30 min) then filtered (0.8 micron, Millipore) before loading onto IMAC resin (Qiagen Superflow FF) at a rate of 1–2 mL/min. The column (∼10 mL) was washed (10 volumes) with IMAC buffer 2 (buffer 1 plus 500 mM NaCl) then with IMAC buffer 3 (buffer 2 plus 65 mM imidazole), before protein elution with IMAC buffer 4 (buffer 2 plus 250 mM imidazole). Usually, 90% of the target was eluted in the first 15–30 mL as assayed by SDS-PAGE. Appropriate fractions were dialyzed overnight into buffer (Tris 20 mM pH 8.0, 150 mM NaCl and 2 mM DTT or β-mercaptoethanol), before the SUMO domain was removed from the N-terminus of 2Apro by incubation with 0.5 mg SUMO protease (prepared in house) for 3–4 h at 30°C. The sample was loaded onto an IMAC column freshly equilibrated with IMAC buffer 1, which bound the His-tagged SUMO domain. The 2Apro target was retrieved in the flow-through (4–5 fractions of 5–10 mL) and pooled. The final fractionation was by gel filtration (GE Healthcare HiPrep 16/60 Sephacryl S-200, 20 mM Tris, pH 8.0, 150 mM NaCl, 2 mM DTT). The purified protein was spin concentrated (Sartorius Vivaspin 20 10 kDa PES concentrator, 5,000 g) and then drop frozen in liquid nitrogen. The final yield was 27.5 mg of purified protein from 0.5 L double-labeled Martek (rich) media. The purity of protein samples was determined by SDS-PAGE (Figure 2). The C105A variant protein aggregated less during purification and produced a higher yield of protein.

Bottom Line: The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues.The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%.Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

View Article: PubMed Central - PubMed

Affiliation: National Magnetic Resonance Facility at Madison, Biochemistry Department, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Human rhinovirus strains differ greatly in their virulence, and this has been correlated with the differing substrate specificity of the respective 2A protease (2Apro). Rhinoviruses use their 2Apro to cleave a spectrum of cellular proteins important to virus replication and anti-host activities. These enzymes share a chymotrypsin-like fold stabilized by a tetra-coordinated zinc ion. The catalytic triad consists of conserved Cys (C105), His (H34), and Asp (D18) residues. We used a semi-automated NMR protocol developed at NMRFAM to determine the solution structure of 2Apro (C105A variant) from an isolate of the clinically important rhinovirus C species (RV-C). The backbone of C2 2Apro superimposed closely (1.41-1.81 Å rmsd) with those of orthologs from RV-A2, coxsackie B4 (CB4), and enterovirus 71 (EV71) having sequence identities between 40% and 60%. Comparison of the structures suggest that the differential functional properties of C2 2Apro stem from its unique surface charge, high proportion of surface aromatics, and sequence surrounding the di-tyrosine flap.

Show MeSH
Related in: MedlinePlus