Foot-and-mouth disease virus 2C is a hexameric AAA+ protein with a coordinated ATP hydrolysis mechanism

Abstract

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.

FIGURE 1.

Modifications of the N terminus of FMDV 2C to enhance soluble overexpression in E. coli. a, schematic representation of the modified FMDV 2C proteins produced for this study. The Walker A (WA), Walker B (WB), and C (MC) motifs are indicated. Residues mutated in these motifs are underlined. The predicted N-terminal amphipathic region is shaded gray; the black box indicates the N-terminal His₆ tag. b, E. coli growth curves following induction of His₆-tagged wild type 2C(1–318) (broken line), the Walker A mutant (K116A) 2C(1–318)WA (dotted line), or the deletion mutant 2C(34–318) (solid line); cell density was monitored by OD measurements at 600 nm. c, size exclusion chromatography of purified FMDV 2C(34–318) and 2C(61–318). In five separate runs, 10 mg (solid line), 7.5 mg (dashed line), 5 mg (dotted line), and 2.5 mg (dashed-single dotted line) of 2C(34–318) or 10 mg of 2C(61–318) (dashed-double dotted line) was loaded onto the column. The elution positions of molecular mass standards are indicated: 1) β-amylase (200 kDa), 2) alcohol dehydrogenase (150 kDa); 3) BSA (66 kDa), 4) carbonic anhydrase (29 kDa), and 5) cytochrome c (12.5 kDa). d, 12% SDS-PAGE of the peak fractions from the 10-mg run shown in c.

FIGURE 2.

FMDV 2C is a specific ATPase that is sensitive to NaCl and GdnHCl. The ATPase activity of FMDV 2C(34–318) was measured in 100-μl reactions containing 4 μg of protein (1.25 μm), 1 mm ATP, and 2 mm MgCl₂, unless otherwise stated. a, ATP hydrolysis was measured in the presence of varying concentrations of protein (open diamonds). There was no detectable ATPase activity for the Walker A, K116A (WA) (open circles), Walker B, D160A (WB) (open triangles), and Motif C, N207A (MC) (crosses). The data plotted are an average of two experiments. b, the ATPase activity is very sensitive to NaCl and decreases monotonically with an ED₅₀ of about 10 mm. c, the ATPase activity of 2C(34–318) varies in a complex manner with increases in ATP concentration, exhibiting a sharp decline between 1.25 and 1.5 mm substrate. d, comparative NTPase activity of 2C(34–318) with 1 mm each ATP, GTP, CTP, and UTP. e, UV cross-linking of [α-³²P]ATP to FMDV 2C(34–318) and various mutants (identified below each lane). After cross-linking, the protein sample was run on a 12% SDS-polyacrylamide gel. Top, the positions of protein bands were confirmed by staining with Coomassie Blue before gel drying; bottom, cross-linked ATP was detected by exposure to a phosphor screen (bottom). 5 mm unlabeled ADP or ATP was included as indicated. f, effect of GdnHCl on the ATPase activity of wild-type FMDV 2C(34–318) (WT) (closed circles) and the 2C(34–318) M159L mutant (open circles). Error bars, S.E. determined from three independent measurements. WA, Walker A; WB, Walker B; MC, motif C.

FIGURE 3.

FMDV 2C binds to ssRNA with higher affinity than to dsRNA. The binding of 2C(34–318) to ³²P-5′-end-labeled RNA was measured in electrophoretic mobility shift assays (see “Experimental Procedures”). Protein-RNA complexes were resolved on a native 10% polyacrylamide gel, which was then exposed to a phosphor screen. The positions of the free unbound probe (Free) and protein-RNA complex (Com) are indicated. Varying concentrations of 2C(34–318) (2C) were added to labeled 20-base ssRNA (5′-AAUUCUAAGGGCCAGCGAGA-3′) (a) or labeled 10-base ssRNA (5′-CCUUAGAAUU-3′) (b). c, a dsRNA probe (DS) was prepared by mixing the 20-base-long probe from a with an unlabeled complementary RNA of exactly the same length, at a molar ratio of 1:1.2, heating to 70 °C for 5 min, and cooling to 22 °C over 2 h. d, the ATPase activity of 2C(34–318) was measured in the presence of various amounts of single-stranded RNA. Error bars, S.E. determined from three independent measurements.

FIGURE 4.

ATP and RNA stimulate the hexamerization of FMDV 2C. Oligomerization of 2C(34–318) and 2C(61–318) was analyzed by SEC and SEC-MALS. 10 mg of 2C(34–318) (a) or 2C(61–318) (b) was loaded onto a Superdex S200 10/30 column coupled to in-line light scattering and refractive index detectors. The refractive index measurement (dotted line) indicates the concentration of protein in the eluate. The molecular weight of the protein species in the eluate (determined from the refractive index and light scattering measurements) (63)) is indicated by the solid line. c–f, 100 μl of 5 mg/ml wild type or mutant 2C(34–318) or 2C(61–318) protein were mixed with 2 mm ATP or ADP, 5 mm MgCl₂, and a 5-fold molar excess of 20-nucleotide ssRNA (as indicated) and incubated for 10 min at 22 °C prior to injection onto a Superdex S200 10/30 column for analysis by standard SEC (c and d) or SEC-MALS (e and f). The arrow in c indicates the sample used for electron microscopy work (see Fig. 5).

FIGURE 5.

The Motif C mutant of FMDV 2C(34–318) forms hexameric rings in the presence of ATP and RNA. A sample of the 2C(34–318) Motif C mutant (N207A) hexamers obtained in the presence of ATP and RNA (indicated by the arrow in Fig. 4c) was applied to glow-discharged carbon grids and negatively stained with uranyl acetate. Electron micrographs were taken on a Philips CM200 electron microscope operating at 200,000 V under low dose conditions of 10 e⁻/Ų. Images were captured directly at 50,000 times magnification over a range of nominal defocus (0.5–2 μm) on a CCD camera. a, a representative micrograph of FMDV 2C particles (indicated by arrows). Scale bar, 200 Å. b, a selection of FMDV 2C particles following initial processing by IMAGIC (54). Scale bar, 100 Å. c, a range of hexameric FMDV 2C class averages (∼20 particles/class). Scale bar, 100 Å. d, left to right, first, second, third, and fourth eigenimages reveal the 6-fold symmetry in the FMDV 2C data set.

FIGURE 6.

ATP hydrolysis by FMDV 2C is coordinated. a, various ratios of wild type 2C(34–318) (WT) and 2C(34–318) Walker A mutant, K116A (MUT), were mixed before addition to an ATPase assay. b, various ratios of wild type 2C(61–318) and 2C(61–318) Walker A mutant, K116A, were mixed before addition to an ATPase assay. Asynchronous ATP hydrolysis would be expected to decrease linearly as the amount of inactive enzyme is increased (dotted line in both plots). Data are averages of three experiments ± S.E. (error bars).