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

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 a K(d) value of 205 μM, and hydrolyses 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 by N- ethylmaleimide. Neither the soluble receptor, HisJ, nor the transport substrate, histidine, has any effect on the activity.