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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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RV sequences by species.WebLogo depictions [46] summarize full species alignment information for key 2Apro residues. RV polyprotein alignments have been described [8]. This dataset compared RV-A (79 types, 208 seqs), RV-B (30 types, 74 seqs), RV-C (32 types, 67 seqs). The residue height indicates the relative amino acid frequency. The A2, B14 and C2 numbering system is for the native, ungapped proteins.
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pone-0097198-g009: RV sequences by species.WebLogo depictions [46] summarize full species alignment information for key 2Apro residues. RV polyprotein alignments have been described [8]. This dataset compared RV-A (79 types, 208 seqs), RV-B (30 types, 74 seqs), RV-C (32 types, 67 seqs). The residue height indicates the relative amino acid frequency. The A2, B14 and C2 numbering system is for the native, ungapped proteins.

Mentions: Another possibility is that the substrate binding pocket, sensitive to the P8−P2′ sequence of the substrate, is the key to specificity [15]. Created in part by the variable di-tyrosine flap, the binding groove is responsive, even during the autocatalytic self-cleaving event, to the sequence and shape of the substrate that fills it. When nine amino acids flanking the NH2-terminus of B14 2Apro were substituted into an A1 or A2 context, the chimeras were unable to cleave themselves from their polyproteins [45]. The same was true when the A2 enzyme was tested in trans against peptides encoding other RV processing sites, even those from closely related viruses [16]. It required at least three substitutions within this length to re-establish activity. The protease reacted to mutated residues in the P2, P1 and P2′ locations during cis reactions [45], but is apparently tolerant of certain changes in the P1, P2′, and P3′ locations during trans reactions [16]. Clearly, all these enzymes are sensing both the shape and sequence of their targets [14]. A WebLogo depiction [46] summarizing all known RV sequences within the self-cleavage sites (Figure 9) highlights the variability encoded here. Not only are the RV-B enzymes extended by two amino acids (cleavage is between positions “−1” and “1”), there is almost no consensus within or between species. The di-tyrosine flap, both upstream and downstream of the few conserved residues (YYP) is another region with pronounced variability. The flap forms one side of the binding cleft (Figure 5B) where substrate acceptance is a prerequisite to the conformational changes that occur during catalysis. In contrast, the zinc-binding residues, the catalytic triad, and C-terminal di-peptide (Q/G) recognized by 3Cpro are absolutely conserved in all species, types, and isolates (n = 348). The 3Cpro enzymes as a rule have more limited selectivity, and for all RV, the carboxyl terminus of 2Apro is released at an identical Gln/Gly pair.


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)

RV sequences by species.WebLogo depictions [46] summarize full species alignment information for key 2Apro residues. RV polyprotein alignments have been described [8]. This dataset compared RV-A (79 types, 208 seqs), RV-B (30 types, 74 seqs), RV-C (32 types, 67 seqs). The residue height indicates the relative amino acid frequency. The A2, B14 and C2 numbering system is for the native, ungapped proteins.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0097198-g009: RV sequences by species.WebLogo depictions [46] summarize full species alignment information for key 2Apro residues. RV polyprotein alignments have been described [8]. This dataset compared RV-A (79 types, 208 seqs), RV-B (30 types, 74 seqs), RV-C (32 types, 67 seqs). The residue height indicates the relative amino acid frequency. The A2, B14 and C2 numbering system is for the native, ungapped proteins.
Mentions: Another possibility is that the substrate binding pocket, sensitive to the P8−P2′ sequence of the substrate, is the key to specificity [15]. Created in part by the variable di-tyrosine flap, the binding groove is responsive, even during the autocatalytic self-cleaving event, to the sequence and shape of the substrate that fills it. When nine amino acids flanking the NH2-terminus of B14 2Apro were substituted into an A1 or A2 context, the chimeras were unable to cleave themselves from their polyproteins [45]. The same was true when the A2 enzyme was tested in trans against peptides encoding other RV processing sites, even those from closely related viruses [16]. It required at least three substitutions within this length to re-establish activity. The protease reacted to mutated residues in the P2, P1 and P2′ locations during cis reactions [45], but is apparently tolerant of certain changes in the P1, P2′, and P3′ locations during trans reactions [16]. Clearly, all these enzymes are sensing both the shape and sequence of their targets [14]. A WebLogo depiction [46] summarizing all known RV sequences within the self-cleavage sites (Figure 9) highlights the variability encoded here. Not only are the RV-B enzymes extended by two amino acids (cleavage is between positions “−1” and “1”), there is almost no consensus within or between species. The di-tyrosine flap, both upstream and downstream of the few conserved residues (YYP) is another region with pronounced variability. The flap forms one side of the binding cleft (Figure 5B) where substrate acceptance is a prerequisite to the conformational changes that occur during catalysis. In contrast, the zinc-binding residues, the catalytic triad, and C-terminal di-peptide (Q/G) recognized by 3Cpro are absolutely conserved in all species, types, and isolates (n = 348). The 3Cpro enzymes as a rule have more limited selectivity, and for all RV, the carboxyl terminus of 2Apro is released at an identical Gln/Gly pair.

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