The Hydrophilic N-terminal Domain Complements the Membrane-anchored C-terminal Domain of the Sensor Kinase KdpD ofEscherichia coli

Abstract

The putative turgor sensor KdpD is characterized by a large, N-terminal domain of about 400 amino acids, which is not found in any other known sensor kinase. Comparison of 12 KdpD sequences from various microorganisms reveals that this part of the kinase is highly conserved and includes two motifs (Walker A and Walker B) that are very similar to the classical ATP-binding sites of ATP-requiring enzymes. By means of photoaffinity labeling with 8-azido-[α-32P]ATP, direct evidence was obtained for the existence of an ATP-binding site located in the N-terminal domain of KdpD. The N-terminal domain, KdpD/1–395, was overproduced and purified. Although predicted to be hydrophilic, it was found to be membrane-associated and could be solubilized either by treatment with buffer of low ionic strength or detergent. The membrane-associated form, but not the solubilized one, retained the ability to bind 8-azido-[α-32P]ATP. Previously, it was shown that the phosphatase activity of a truncated KdpD, KdpD/Δ12–395, is deregulated in vitro (Jung, K., and Altendorf, K. (1998)J. Biol. Chem. 273, 17406–17410). Here, we demonstrated that this effect was reversed in vesicles containing both the truncated KdpD and the N-terminal domain. Furthermore, coexpression of kdpD/Δ12–395 and kdpD/1–395 restored signal transduction in vivo. These results highlight the importance of the N-terminal domain for the function of KdpD and provide evidence for an interaction of this domain and the transmitter domain of the sensor kinase. The putative turgor sensor KdpD is characterized by a large, N-terminal domain of about 400 amino acids, which is not found in any other known sensor kinase. Comparison of 12 KdpD sequences from various microorganisms reveals that this part of the kinase is highly conserved and includes two motifs (Walker A and Walker B) that are very similar to the classical ATP-binding sites of ATP-requiring enzymes. By means of photoaffinity labeling with 8-azido-[α-32P]ATP, direct evidence was obtained for the existence of an ATP-binding site located in the N-terminal domain of KdpD. The N-terminal domain, KdpD/1–395, was overproduced and purified. Although predicted to be hydrophilic, it was found to be membrane-associated and could be solubilized either by treatment with buffer of low ionic strength or detergent. The membrane-associated form, but not the solubilized one, retained the ability to bind 8-azido-[α-32P]ATP. Previously, it was shown that the phosphatase activity of a truncated KdpD, KdpD/Δ12–395, is deregulated in vitro (Jung, K., and Altendorf, K. (1998)J. Biol. Chem. 273, 17406–17410). Here, we demonstrated that this effect was reversed in vesicles containing both the truncated KdpD and the N-terminal domain. Furthermore, coexpression of kdpD/Δ12–395 and kdpD/1–395 restored signal transduction in vivo. These results highlight the importance of the N-terminal domain for the function of KdpD and provide evidence for an interaction of this domain and the transmitter domain of the sensor kinase. transmembrane domains 1 through 4 polymerase chain reaction polyacrylamide gel electrophoresis, LDAO, lauryldimethylamine oxide adenosine 5′-O-(thiotriphosphate) The membrane-bound sensor kinase KdpD and the soluble response regulator KdpE comprise a sensor kinase/response regulator system ofEscherichia coli, which regulates expression of thekdpFABC operon (1.Walderhaug M.O. Polarek J.W. Voelkner P. Daniel J.M. Hesse J.E. Altendorf K. Epstein W. J. Bacteriol. 1992; 174: 2152-2159Crossref PubMed Google Scholar) encoding the high affinity K+-translocating complex KdpFABC (see Ref. 2.Altendorf K. Epstein W. Lee A.G. Biomembranes. 5. JAI Press Inc., London1996: 403-420Google Scholar for review). KdpD consists of a long hydrophilic N-terminal domain, four putative transmembrane domains (TM1–TM4),1 and an extended C-terminal transmitter domain (see Fig. 1) (3.Zimmann P. Puppe W. Altendorf K. J. Biol. Chem. 1995; 270: 28282-28288Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). KdpD undergoes autophosphorylation at a histidine residue in the presence of ATP, and subsequently, the phosphoryl group is transferred to the response regulator KdpE (4.Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar, 5.Nakashima K. Sugiura A. Momoi H. Mizuno T. Mol. Microbiol. 1992; 6: 1777-1784Crossref PubMed Scopus (53) Google Scholar). Phosphorylated KdpE exhibits an increased affinity to bind upstream of the kdpFABC promoter region and consequently triggers transcription of this operon (6.Sugiura A. Nakashima K. Tanaka K. Mizuno T. Mol. Microbiol. 1992; 6: 1769-1776Crossref PubMed Scopus (64) Google Scholar). The enzymatic activities of KdpD have been shown with purified protein reconstituted into proteoliposomes and purified KdpE (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Moreover, KdpD is found to be the only protein that mediates the dephosphorylation of purified KdpE∼P (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recently, we have shown that KdpD is a homodimer and that there is no change of the oligomeric state upon phosphorylation (8.Heermann R. Altendorf K. Jung K. Biochim. Biophys. Acta. 1998; 1415: 114-124Crossref PubMed Scopus (30) Google Scholar). Based on studies with KdpD proteins, in which different Arg residues were individually replaced with Gln, an electrostatic switch mechanism within the protein is proposed, which regulates the ratio of kinase to phosphatase activity (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The concept of this is supported by the fact that KdpD kinase activity is dependent on negatively charged phospholipids (10.Stallkamp I. Dowhan W. Altendorf K. Jung K. Arch. Microbiol. 1999; 172: 295-302Crossref PubMed Scopus (22) Google Scholar). The stimulus, which KdpD senses, is believed to be a decrease in turgor or an effect thereof. Expression of kdpFABC is induced under K+-limiting growth conditions (less than 2 mm), where the constitutive K+-translocating systems TrkG, TrkH, and Kup of E. coli are unable to maintain the required intracellular pool of K+. In mutants lacking these K+ transport systems, kdpFABC is already expressed in media containing less than 50 mmK+. Therefore, KdpD seems to sense the cell's “need” for K+ to maintain turgor (11.Laimins L.A. Rhoads D.B. Epstein W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 464-468Crossref PubMed Scopus (189) Google Scholar, 12.Epstein W. Acta Physiol. Scand. Suppl. 1992; 607: 193-199PubMed Google Scholar, 13.Malli R. Epstein W. J. Bacteriol. 1998; 180: 5102-5108Crossref PubMed Google Scholar). Control by turgor is supported by the finding that a sudden increase in medium osmolarity, which reduces turgor, transiently turns on kdpFABCexpression. This model has been challenged by the finding that under some conditions expression of kdpFABC is only significantly induced when the osmolarity of the medium is increased by salt and not in case of sugar (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 14.Sutherland L. Cairney J. Elmore M.J. Booth I.R. Higgins C.F. J. Bacteriol. 1986; 168: 805-814Crossref PubMed Google Scholar, 15.Asha H. Gowrishankar J. J. Bacteriol. 1993; 175: 4528-4537Crossref PubMed Google Scholar). Based on kdpD mutants, which give rise to K+-independent kdpFABC expression but regain the ability to respond to changes in medium osmolarity independently of the solute, it is suggested that KdpD senses two stimuli: increase in osmolarity and K+ limitation (16.Sugiura A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar). This presumption is supported by the differentiated response of KdpD proteins with individual substitutions of Arg residues toward K+ limitation and osmotic upshock (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Compared with other known sensor kinases, KdpD is the only protein that has a large, hydrophilic N-terminal domain of about 400 amino acids in length (17.Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig. 1). Sequence comparison of 12 KdpD sequences of various bacteria reveals that this domain is more conserved then other regions of KdpD. This N-terminal domain includes two sequence motifs, which are very similar to those of a typical ATP-binding site (Walker A and B motifs) (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (Fig.2). Sequences of short N-terminal versions of KdpD have been found in Synechocystis sp. (19.Kaneko T. Sato S. Kotani H. Tanaka A. Asamizu E. Nakamura Y. Miyajima N. Hirosawa M. Sugiura M. Sasamoto S. Kimura T. Hosouchi T. Matsuno A. Muraki A. Nakazaki N. Naruo K. Okumura S. Shimpo S. Takeuchi C. Wada T. Watanabe A. Yamada M. Yasuda M. Tabata S. DNA Res. 1996; 3: 109-136Crossref PubMed Scopus (2111) Google Scholar) and in Anabaena sp. L-31 (GenBank™ accession numberAF213466). It is suggested that such a fragment may form a functional KdpD complex with a C-terminal fragment that includes the membrane-spanning domains. It has been shown recently that the phosphatase activity of KdpD is significantly increased in the presence of ATP or nonhydrolyzable ATP analogues, whereas other nucleotides have no effect. Truncated KdpD proteins lacking different parts of the N-terminal domain, including amino acids 12–128 are characterized by phosphatase activities independent of the presence of ATP. This finding suggests that an ATP-binding site has a regulatory role within this domain (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here, we show that the N-terminal domain of E. coli KdpD can be produced separately allowing both the functional and biochemical characterization of this domain. [γ-32P]ATP, NAP-5 gel filtration columns and thrombin were purchased from Amersham Pharmacia Biotech. 8-Azido-[α-32P]ATP was from ICN Biomedicals. Goat anti-(rabbit IgG)-alkaline phosphatase and anti-(mouse IgG)-alkaline phosphatase conjugates were purchased from Biomol. Ni-NTA agarose and penta-His antibody were from Qiagen. All other reagents were reagent grade and obtained from commercial sources. E. coli strain JM 109 (recA1 endA1 gyrA96 thi hsdR17 supE44λ− relA1Δ(lac-proAB)/F′ traD36 proA + B + lacIqlacZΔM15) (20.Yanisch-Perron C. Vieira J. Messing J. Gene. 1985; 33: 103-119Crossref PubMed Scopus (11449) Google Scholar) was used as carrier for the plasmids described. E. coli strains TK2240 (kdp + trkA405 trkD1) and TKW22692 (kdpD6-92 trkA405 trkD1) (17.Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) were used for complementation of kdpD/1–395 with kdpD/Δ12–395. E. coli strain TKR2000 (ΔkdpFABCDE trkA405 trkD1 atp706) (21.Kollmann R. Altendorf K. Biochim. Biophys. Acta. 1993; 1143: 62-66Crossref PubMed Scopus (32) Google Scholar) harboring plasmid pPV5–1 (22.Jung K. Heermann R. Meyer M. Altendorf K. Biochim. Biophys. Acta. 1998; 1372: 311-322Crossref PubMed Scopus (19) Google Scholar) or pPV5-3 (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and its derivatives carrying different kdpD mutations was used for expression ofkdpD from the tac promoter. Plasmid pWP6-92 carries an in-frame deletion of 852 encoding (17.Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Plasmid are as the amino is used by a the of the residue in the KdpD. The the amino at this (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In plasmid kdpD was into M.J. J. J. Bacteriol. 1995; PubMed Scopus Google in which expression of kdpD is under of the promoter (22.Jung K. Heermann R. Meyer M. Altendorf K. Biochim. Biophys. Acta. 1998; 1372: 311-322Crossref PubMed Scopus (19) Google Scholar). In plasmid (8.Heermann R. Altendorf K. Jung K. Biochim. Biophys. Acta. 1998; 1415: 114-124Crossref PubMed Scopus (30) Google kdpD was into M.J. J. J. Bacteriol. 1995; PubMed Scopus Google Scholar). Plasmid is with plasmids and and can be in E. of a thrombin site amino in KdpD was by individual amino kdpD/1–395 were obtained by of or for and the to amino All mutations were by the polymerase chain reaction Gene. PubMed Scopus Google Scholar). were purified in agarose and with enzymes. DNA were from the gel and to (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) or pPV5-3 in plasmids and and were obtained by of plasmids and with and the were with a E. coli strain TKR2000 with plasmids or carrying different kdpD mutations was at in complex medium and with or with and of under of the promoter was by of to the were at an at of vesicles were as (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) with the that vesicles containing the N-terminal domain were not with buffer of low ionic were in 50 containing in and at and of and were as for KdpD (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). with lauryldimethylamine oxide and at for the protein was to the with buffer by of the solubilized protein with for at 4 the N-terminal domain, was purified in the of detergent. this vesicles were four with buffer of low ionic strength at for was found in the soluble and were the protein was on a which was with buffer detergent. The complex was then into a and protein was by with KdpD derivatives were by the to was reconstituted into E. coli phospholipids as (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). was purified and reconstituted into proteoliposomes as of in buffer and 2 were with of for at was by with at for thrombin and were and was extended for at vesicles containing in 50 and protein 1 were with of for at and was photoaffinity was purified as the was in reaction of the of agarose was by at All were to gel PubMed Scopus Google Scholar). were ATP was by of the to a and were the system that of protein were were with E. coli strains with plasmids and and TK2240 were in medium W. M. J. Bacteriol. PubMed Google Scholar) containing K+ to an at of A sudden decrease of K+ was by medium K+ in K+ of were by and in buffer PubMed Scopus Google Scholar). and KdpFABC was was obtained as (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). was by of vesicles containing KdpD derivatives when and different were and the reaction was by of buffer PubMed Scopus Google Scholar). All were to an was on the were and phosphorylation of KdpE was and as was by a Biochem. PubMed Scopus Google as a of KdpD, and proteins was with KdpD and and penta-His to a (4.Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). A short of KdpD the N-terminal domain acids by residues was by of for and at the site in This was in E. coli strain which was with plasmid not studies that was membrane-associated (Fig. of the protein was found in the (Fig. could either be solubilized by not or from the under low ionic conditions 4 and (see obtained by both was used for in Fig. either in the presence or of to Ni-NTA agarose and can be from the by the to under low ionic buffer conditions only membrane-associated proteins are of about whereas in the presence of two proteins are (Fig. of by Ni-NTA affinity was as under is a solubilized proteins protein as 1 and with protein in with protein protein was from the under low ionic buffer conditions The N-terminal domain is highly conserved known KdpD sequences of various Moreover, it a highly conserved which is very similar to a classical ATP-binding site (Walker A and Walker B motifs) (Fig. biochemical evidence for of ATP to this site was obtained by photoaffinity labeling with 8-azido-[α-32P]ATP. The purified and reconstituted length sensor carrying a thrombin site at amino to the ATP-binding sites of the N-terminal and C-terminal domain, was with and then with The protein were on an and the was (see the and KdpD, two were not to the fragment thrombin of the N-terminal sequences of these that the fragment amino acids and the ATP-binding site within the N-terminal domain of KdpD. The of protein of the fragment was low to the N-terminal the amino of this fragment is The was of the protein with an of ATP. shown in Fig. length KdpD but no were the for of ATP. of KdpD, in which the ATP-binding site was was the only with that of KdpD not shown in Fig. the N-terminal domain the ability to bind when it is In no labeling of the solubilized N-terminal domain was by treatment with buffer The importance of the N-terminal domain for the function of the sensor kinase was shown by complementation as under this coli strain TKW22692 (17.Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) carrying an in-frame deletion within kdpD encoding was expression was by the transport a only is (Fig. In of which were on a of thekdpFABC and (17.Puppe W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google it is found that KdpFABC is in this strain in comparison to a strain (Fig. 1 and of E. coli TKW22692 with plasmid encoding the N-terminal domain of KdpD, KdpD/1–395, rise to a increase in the of produced KdpFABC complex (Fig. The effect was for the N-terminal domain, that the not complementation not no increase in KdpFABC was when the ATP-binding site in the N-terminal domain was by amino (Fig. These results the importance of the ATP-binding site in the hydrophilic N-terminal domain of KdpD and provide evidence for the of an interaction of this domain with the of the The sensor kinase KdpD is characterized by two enzymatic kinase and phosphatase In phosphatase activity is high in the presence of ATP or nonhydrolyzable ATP analogues, whereas in the of ATP dephosphorylation of KdpE∼P is very KdpD lacking the ATP-binding site of the N-terminal domain or by are characterized by a phosphatase activity that is independent of the presence of ATP (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google the N-terminal domain can the truncated KdpD in plasmids encoding KdpD/Δ12–395, and encoding the N-terminal domain of KdpD, were in E. coli strain were the two kdpD derivatives were and KdpD phosphatase activity was of shown in Fig. vesicles containing both the truncated KdpD as as the N-terminal domain are characterized by a KdpE∼P phosphatase which a restored on ATP. These the of a direct interaction of the N-terminal domain with the of the protein and the importance of the ATP-binding site located in this The that KdpD senses to signal which results in of kdpFABC is believed to be a decrease in turgor or some effect the role of K+ as an W. Acta Physiol. Scand. Suppl. 1992; 607: 193-199PubMed Google Scholar). there is an as to KdpD is a turgor sensor or not R. Epstein W. J. Bacteriol. 1998; 180: 5102-5108Crossref PubMed Google Scholar, 14.Sutherland L. Cairney J. Elmore M.J. Booth I.R. Higgins C.F. J. Bacteriol. 1986; 168: 805-814Crossref PubMed Google Scholar, 15.Asha H. Gowrishankar J. J. Bacteriol. 1993; 175: 4528-4537Crossref PubMed Google Scholar, A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar). the of the for KdpD is Although KdpD high to transmitter domains of other histidine kinases, it is in of the domain. This domain an extended N-terminal four transmembrane and part of the C-terminal domain (Fig. 1). regions within the domain have been to be for the activity of A of charged Arg residues to transmembrane domain four is in the kinase and phosphatase activities of KdpD (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The four transmembrane domains to be for the of and C-terminal domains to other as as for signal transduction (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, W. Zimmann P. Jung K. Lucassen M. Altendorf K. J. Biol. Chem. 1996; 271: 25027-25034Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, K. Heermann R. Meyer M. Altendorf K. Biochim. Biophys. Acta. 1998; 1372: 311-322Crossref PubMed Scopus (19) Google Scholar). acids including a Walker A to be for the of the phosphatase activity of KdpD (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here, we the and of the N-terminal domain of KdpD as as its functional Although the Walker A found in the N-terminal domain of KdpD is different from a classical amino is found the and photoaffinity labeling by shown direct evidence that the N-terminal domain ATP. This has been shown for the length sensor kinase and for the separately produced domain. The other known for the of the Walker A is found in the W. J. Y. 1995; PubMed Scopus (56) Google for which ATP was ATP was only for the membrane-associated N-terminal domain, but not for the solubilized Although the N-terminal domain is of hydrophilic amino there is a of amino residues the Walker A site (3.Zimmann P. Puppe W. Altendorf K. J. Biol. Chem. 1995; 270: 28282-28288Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). the found in the ability to bind it is that only the membrane-associated domain the to the It has been shown for the ATP which is by intracellular ATP, that affinity changes significantly on the 1998; PubMed Scopus Google Scholar). negatively charged phospholipids the kinase activity of KdpD (10.Stallkamp I. Dowhan W. Altendorf K. Jung K. Arch. Microbiol. 1999; 172: 295-302Crossref PubMed Scopus (22) Google it is that in the of this domain to the the to ATP. The truncated KdpD proteins and but not kdpFABC expression significantly under K+-limiting growth conditions (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). these are the only known of KdpD which the response to K+ Although other amino within KdpD the response to osmolarity to an increase in the K+ of the response to K+ limitation (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). In the we have shown that expression of the N-terminal domain significantly kdpFABC expression under K+-limiting growth This the importance of this domain for signal transduction and evidence for an interaction the N-terminal domain and the C-terminal transmitter domain of KdpD. It has been that of amino acids 12–128 of the phosphatase activity in In the presence of ATP or nonhydrolyzable ATP analogues, dephosphorylation of KdpE∼P is whereas in the of ATP only a very is (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Truncated KdpD in which amino acids 12–128 are are characterized by an phosphatase activity (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Here, we demonstrated that an ATP of the dephosphorylation of KdpE∼P by is restored in the presence of the separately produced N-terminal domain. The role of the ATP-binding site within the N-terminal domain is under conditions of an osmotic upshock T. S. Arch. Biochem. Biophys. PubMed Scopus (56) Google Scholar) as as under K+-limiting growth conditions a sudden increase of the intracellular ATP is ATP the phosphatase activity of KdpD, a role of the intracellular ATP as a for KdpD to the ratio kinase and phosphatase activities is the direct the increased intracellular ATP and of ATP to the N-terminal domain is In the results that the N-terminal domain is for the phosphatase activity of KdpD and signal The provide direct evidence for the existence of an ATP-binding site within this domain. The role of this site KdpD activity can be as as in vitro from two separately produced domains evidence for the interaction the N-terminal domain and the of the the for the N-terminal domain the to this interaction in more for N-terminal of KdpD and for of for of the