Purification and Characterization of HisP, the ATP-binding Subunit of a Traffic ATPase (ABC Transporter), the Histidine Permease of Salmonella typhimurium

doi:10.1074/jbc.272.44.27745

Purification and Characterization of HisP, the ATP-binding Subunit of a Traffic ATPase (ABC Transporter), the Histidine Permease of Salmonella typhimurium

SOLUBILITY, DIMERIZATION, AND ATPase ACTIVITY*

From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202

Abstract

The nucleotide-binding subunit, HisP, of the histidine permease, a traffic ATPase (ABC transporter), has been purified as a soluble protein and characterized. Addition of a 6-histidine extension (HisP_(His6)) allows a rapid and effective metal affinity purification, giving a 30-fold purification with a yield of 50%. HisP_(his6) is indistinguishable from underivatized HisP when incorporated into the permease membrane-bound complex, HisQMP₂. Purified HisP_(his6) has a strong tendency to precipitate; 5 mm ATP and 20% glycerol maintain it in solution at a high protein concentration. HisP_(his6) is active as a dimer, binds ATP with aK _d value of 205 μm, and hydrolyzes it at a rate comparable to that of HisQMP₂; in contrast to the latter, it does not display cooperativity for ATP. HisP_(his6) has been characterized with respect to substrate and inhibitor specificity and various physico-chemical characteristics. Its pH optimum is 7 and it requires a cation for activity, with Co²⁺ and Mn²⁺ being more effective than Mg²⁺ at lower concentrations but inhibitory in the higher concentration range. In contrast to the intact complex, HisP_(his6) is not inhibited by vanadate but is inhibited byN-ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.

Footnotes

↵* This work was supported by National Institutes of Health Grant DK12121 (to G. F-L. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: 510-642-1979; Fax: 510-643-7935.
↵1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; HisQMP₂, the membrane-bound complex containing HisQ, HisM, and HisP; HisP_(his6), HisP protein with a carboxyl-terminal extension of 8 amino acid residues: Leu-Glu-His-His-His-His-His-His; PAGE, polyacrylamide gel electrophoresis; TNP-ATP, 2′-(or -3′)-O-(trinitrophenyl)adenosine 5′-triphosphate; PE, phosphatidylethanolamine; NEM, N-ethylmaleimide; IPTG, isopropylthiogalactoside; AMP-PNP, 5′-adenylyl-imidodiphosphate; AMP-PCP, adenylyl (β,γ-methylene)-diphosphonate; ATPγS, adenosine 5′-O-(3-thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography.
↵2 Plasmid pFA284 carries the hisQ, hisM, and hisP _(his6) genes under the temperature-sensitive control of the lambda P_L promoter (strain GA500; laboratory strain collection).
↵3 To exclude the possibility that this material did not stick to the column because of an overloading effect, it was reapplied to a new TALON column. None of it was retained. Therefore, it is assumed that this material has undergone some form of denaturation.
↵4 A small amount of HisP_(his6) (11%) is retained by the column after elution.
↵5 However, exhaustive removal of Mg²⁺by the addition of EDTA in several column washing steps before elution with 100 mm imidazole results in the disappearance of HisP_(his6) from all column fractions (presumably it precipitates within the column matrix).
↵6 That this activity is due to HisP_(his6) rather than to a contaminating ATPase is demonstrated by the fact that preparations obtained by an identical purification scheme from several hisP mutant strains defective in transport do not display any ATPase activity.
↵7 Co²⁺ may stimulate the activity by interacting with the (His₆) tail and inducing a conformational change.
↵8 The membrane-bound complex of the maltose permease, which contains two copies of the ATP-binding subunit, MalK, also displays positive cooperativity for ATP (26).
↵9 This measurement requires the elimination of the ATP obligatorily present in solutions of HisP_(his6) at high concentrations. The ATP analog, TNP-ATP, which has a high affinity for the enzyme and is hydrolyzed very slowly, maintains high concentrations of HisP_(his6) in solution when present at 10 μm, for 14 days at 4 °C. HisP_(his6) was purified as usual and the ATP was replaced with TNP-ATP by passage through a gel filtration column containing 10 μm TNP-ATP. This concentration of TNP-ATP does not significantly interfere with the assay for ATPase activity (Table II).
↵10 G. F.-L. Ames and K. Nikaido, unpublished data.
↵11 The molarity of phospholipids was calculated using 700 as an average molecular weight.
↵12 That this would be so had already been deduced from the higher sensitivity to proteolytic degradation of HisP produced in the absence of the hydrophobic subunits. Although HisP can exist in a soluble form and its sequence gives no indication of hydrophobicity, it has a strong tendency to associate with the membrane (9, 32).
↵13 G. F.-L. Ames and K. Nikaido, unpublished results.
↵14 P.-Q. Liu and G. F.-L. Ames, submitted for publication.
↵15 G. F.-L. Ames and K. Nikaido, unpublished data.
↵16 The inhibition of HisP_(his6) by total phospholipids can be fully accounted for by the inhibitory effects of phosphatidylglycerol and cardiolipin, which, after PE, are the most abundant phospholipids in E. coli (about 25% of the total phospholipids (37)).
- Received July 8, 1997.
- Revision received August 28, 1997.