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. 1998 Mar;180(6):1347-53.
doi: 10.1128/JB.180.6.1347-1353.1998.

When an ATPase is not an ATPase: at low temperatures the C-terminal domain of the ABC transporter CvaB is a GTPase

X Zhong  1 , P C Tai
Affiliations

When an ATPase is not an ATPase: at low temperatures the C-terminal domain of the ABC transporter CvaB is a GTPase

X Zhong et al. J Bacteriol. 1998 Mar.

Abstract

The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins which share a common function and a common nucleotide-binding domain. The CvaB protein from Escherichia coli is a member of the bacterial ABC exporter subfamily and is essential for the export of the peptide antibiotic colicin V. Here we report that, surprisingly, the CvaB carboxyl-terminal nucleotide-binding domain (BCTD) can be preferentially cross-linked to GTP but not to ATP at low temperatures. The cross-linking is Mg2+ and Mn2+ dependent. However, BCTD possesses similar GTPase and ATPase activities at 37 degrees C, with the same kinetic parameters and with similar responses to inhibitors. Moreover, a point mutation (D654H) in CvaB that completely abolishes colicin V secretion severely impairs both GTPase and ATPase activities in the corresponding BCTD, indicating that the two activities are from the same enzyme. Interestingly, hydrolysis activity of ATP is much more cold sensitive than that of GTP: BCTD possesses mainly GTP hydrolysis activity at 10 degrees C, consistent with the cross-linking results. These findings suggest a novel mechanism for an ABC protein-mediated transport with specificity for GTP hydrolysis.

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Figures

FIG. 1
FIG. 1
BCTD can be cross-linked to [α-32P]GTP in much higher affinity than to [α-32P]ATP. (A) Left, Coomassie blue-stained SDS-gel showing protein profiles used in the cross-linking. The molecular size markers are bovine serum albumin (66 kDa [66K]), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). Right, autoradiogram of UV cross-linking with [α-32P]ATP and [α-32P]GTP at 0°C. (B) Competitive binding of GTP to GST-BCTD. GST-BCTD was irradiated with UV (254 nm) in the presence of [α-32P]GTP and Mg2+. The unlabeled nucleotides as indicated (100 μM) were added to the complete reaction mixtures before cross-linking. (C) Left, Coomassie blue-stained gel of His6-BCTD; right, autoradiogram of UV cross-linking with [α-32P]GTP (lane 1) and [α-32P]ATP (lane 2).
FIG. 2
FIG. 2
GTPase and ATPase activities of His6-BCTD at 37°C. (A) Copurification of His6-BCTD with nucleotide hydrolysis activities. The His6-BCTD preparation was fractionated on a calibrated FPLC Superdex-200 gel filtration column (Pharmacia; 0.5 ml min−1) in 50 mM Tris-HCl (pH 7.6)–20 mM KCl–20 mM NH4Cl–1 mM DTT, and fractions of 40 μl were assayed for activity. Size standards included carbonic anhydrase (29 kDa [29KD]), bovine serum albumin (66 kDa), and alcohol dehydrogenase (150 kDa). (B) Substrate concentration titration of GTP and ATP hydrolysis of His6-BCTD at 37°C. (C) Lineweaver-Burk plot of the GTPase and ATPase activities.
FIG. 3
FIG. 3
Effects of point mutation D654H. (A) Effect on ColV secretion. Wild-type (Wt) cvaB strain DH5α(pHK11-4, pXZ11) and D654H mutant DH5α(pHK11-4, pXZ15) were spotted for the ColV assay. (B) Effect on the GTP binding of His6-BCTD. His6-BCTD (wild type and D654H) was subjected to UV cross-linking with [α-32P]ATP and [α-32P]GTP. (C) Effects on GTPase and ATPase activities of His6-BCTD. His6-BCTD (wild type and D654H) was assayed for nucleotide hydrolysis activities in the presence of Mg2+ or Mn2+.
FIG. 4
FIG. 4
Temperature effects of nucleotide cross-linking and hydrolysis. (A) The upper panel is the Coomassie blue-stained gel of protein samples used for the lower panel, which is the autoradiogram of [α-32P]ATP and [α-32P]GTP cross-linkings at different temperatures. (B) Nucleotide hydrolysis activities at different temperatures. Data are means ± standard errors (bars) (n = 4). The GTPase/ATPase ratio at each temperature is shown in the insert.
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