Journal article
Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism
Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N-terminal membrane-binding amphipathic helix attached to the main ATPase domain.
In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34–318), that has ATPase and RNA binding activity.
ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers.
Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism of ATP hydrolysis.
Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection.
Language: | English |
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Publisher: | American Society for Biochemistry and Molecular Biology |
Year: | 2010 |
Pages: | 24347-59 |
ISSN: | 1083351x , 00219258 and 10678816 |
Types: | Journal article |
DOI: | 10.1074/jbc.M110.129940 |
ORCIDs: | Belsham, Graham |
2C protein, viral AAA+ Protein ATPases Adenosine Triphosphate Adenosine Triphosphate/chemistry Amino Acid Motifs Carrier Proteins Carrier Proteins/chemistry Catalytic Domain Enzyme Mechanisms Escherichia coli Escherichia coli/metabolism Foot-and-Mouth Disease Virus Foot-and-Mouth Disease Virus/metabolism Foot-and-mouth Disease Virus Hydrolysis Kinetics Models, Biological Mutation Positive Strand RNA Viruses Protein Assembly Protein Binding Protein Oligomerization Protein Structure, Tertiary RNA RNA, Viral RNA, Viral/metabolism RNA-binding Protein RNA/chemistry Viral Nonstructural Proteins Viral Nonstructural Proteins/chemistry Viral Protein Viral Proteins Viral Proteins/chemistry Viral Replication