FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1990391674> ?p ?o ?g. }

• W1990391674 endingPage "17085" @default.
• W1990391674 startingPage "17080" @default.
• W1990391674 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 base pairs, encoding KdpD/Δ12–395 (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 pPV5-1/G37A, K38A, T39C 2Amino acid replacements are designated as follows: the one-letter amino acid code is used followed by a number indicating the position of the residue in the wild-type KdpD. The second letter denotes the amino acid replacement at this position. encodes KdpD/G37A, K38A, T39C (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). In plasmid pBD, kdpD was cloned into pBAD18 (23.Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3923) Google Scholar), in which expression of kdpD is under control of the arabinose promoter (22.Jung K. Heermann R. Meyer M. Altendorf K. Biochim. Biophys. Acta. 1998; 1372: 311-322Crossref PubMed Scopus (19) Google Scholar). In plasmid pBD3 (8.Heermann R. Altendorf K. Jung K. Biochim. Biophys. Acta. 1998; 1415: 114-124Crossref PubMed Scopus (30) Google Scholar), kdpD was cloned into pBAD33 (23.Guzman L.M. Belin D. Carson M.J. Beckwith J. J. Bacteriol. 1995; 177: 4121-4130Crossref PubMed Scopus (3923) Google Scholar). Plasmid pBD3 is compatible with plasmids pPV5-1 and pBD, and cloned genes can be coexpressed in E. coli. Introduction of a thrombin cleavage site following amino acid 395 in KdpD (KdpD/Th) was achieved by individual amino acid substitutions. Constructs kdpD/1–395 andkdpD/1–395(6His) were obtained by insertion of three stop codons or six codons for His and three stop codons, respectively, after the triplet corresponding to amino acid 395. All site-specific mutations were directed by synthetic mutagenic oligonucleotide primers using the polymerase chain reaction (PCR) overlap extension method (24.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6810) Google Scholar). PCR products were purified in agarose gels and digested with appropriate restriction enzymes. DNA fragments were isolated from the gel and ligated to similarly treated pPV5(6His) (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 resulting in plasmids pPV5-3/Th(6His), pPV5-3/1–395 and pPV5-3/1–395(6His), respectively. Plasmids pPV5-3/1–395/G37A, K38A, T39C and pPV5-3/1–395/G37A, K38A, T39A(6His) were obtained by restriction of plasmids pPV5-3/1–395 and pPV5-3/1–395(6His) with SacI andStuI, and the resulting fragments were ligated with a similarly treated vector, pPV5-1/G37A, K38A, T39C. E. coli strain TKR2000 transformed with plasmids pPV5-3, pBD, or pBD3 carrying different kdpD mutations was grown aerobically at 37 °C in KML complex medium (1% tryptone, 0.5% yeast extract, and 1% KCl) supplemented with ampicillin (100 μg/ml) or with chloramphenicol (34 μg/ml) and ampicillin (100 μg/ml). Overexpression of genes under control of the arabinose promoter was achieved by addition of 0.2% arabinose to the medium. Cells were harvested at an absorbance at 600 nm of ∼1.0. Inverted membrane vesicles were prepared as described previously (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 exception that vesicles containing the N-terminal domain were not washed with buffer of low ionic strength. Vesicles were resuspended in 50 mm Tris-HCl, pH 7.5, containing 10% (v/v) glycerol, frozen in liquid nitrogen, and stored until use at −80 °C. Solubilization and purification of KdpD/1–395(6His) and KdpD/Th(6His) were carried out as described for wild-type 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). Briefly, after solubilization with 2% (v/v) lauryldimethylamine oxide (LDAO) and centrifugation at 100,000 ×g for 30 min, the protein was bound batchwise to the resin [equilibrated with column buffer (50 mm Tris-HCl, pH 7.5; 10% (v/v) glycerol; 0.5 m NaCl; 10 mmβ-mercaptoethanol; 0.04% (w/v) dodecyl maltoside; 10 mmimidazole)] by incubation of the solubilized protein with Ni2+-NTA resin for 30 min at 4 °C. Alternatively, the N-terminal domain, KdpD/1–395(6His), was purified in the absence of detergent. For this purpose, membrane vesicles were washed four times with buffer of low ionic strength (1 mm Tris-HCl, pH 7.5; 3 mm EDTA). After centrifugation at 100,000 ×g for 30 min, KdpD/1–395(6His) was found in the soluble fraction. 49 mm Tris-HCl, pH 7.5, 10% (v/v) glycerol, and 0.5 m NaCl were added before the protein was loaded on a Ni2+-NTA column, which was equilibrated with column buffer without detergent. The protein-resin complex was then packed into a column, and unbound protein was removed by washing with column buffer. Bound His-tagged KdpD derivatives were eluted by increasing the imidazole concentration up to 100 mm. Purified KdpD/Th(6His) was reconstituted into E. coli phospholipids essentially as described (7.Jung K. Tjaden B. Altendorf K. J. Biol. Chem. 1997; 272: 10847-10852Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). KdpD/Th(6His) was purified and reconstituted into proteoliposomes as described above. Proteoliposomes (10 μg of protein) in buffer (50 mm Tris-HCl, pH 7.5; 10% (v/v) glycerol; 10 mm MgCl2; and 2 mm dithiothreitol) were incubated with 5 μCi of 8-azido-[α-32P]ATP (20 Ci/mmol) for 5 min at room temperature. Cross-linking was achieved by irradiating with UV light at 302 nm for 0.5 min. Then, thrombin (5 units) and phosphate-buffered saline were added and incubation was extended for 3 h at room temperature. Inverted membrane vesicles containing KdpD/1–395(6His) (10 mg/ml) in 50 mm Tris-HCl, pH 7.5, 10% (v/v) glycerol, and 10 mm MgCl2 (total protein concentration, 1 mg) were incubated with 25 μCi of 8-azido-[α-32P]ATP (20 Ci/mmol) for 5 min at room temperature, and UV cross-linking was carried out. After photoaffinity labeling, KdpD/1–395(6His) was purified essentially as described above; however, the whole procedure was done in batch using 1.5-ml reaction tubes, because of the small amounts of protein; Ni2+-NTA agarose was separated by centrifugation at 2,000 × g. All samples were immediately subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar). Gels were dried, ATP binding was detected by exposure of the gels to a phosphor screen, and images were analyzed using the PhosphorImager system (Molecular Dynamics). To confirm that equal amounts of protein were used, gels were stained with Coomassie Blue. E. coli strains TKW22692, transformed with plasmids pBD/1–395 and pBD/1–395/G37A, K38A, T39C, respectively, and TK2240 were grown in minimal medium (25.Epstein W. Davies M. J. Bacteriol. 1970; 101: 836-843Crossref PubMed Google Scholar) containing 80 mm K+ to an optical density at 600 nm of ∼1.5. A sudden decrease of K+ was achieved by adding minimal medium without K+ resulting in 20 mm K+ (final concentration). After 30 min of further incubation, cells were harvested by centrifugation and resuspended in SDS sample buffer (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar). After SDS-PAGE and Western blotting, KdpFABC production was detected using antibodies against KdpFABC. KdpE∼32P was obtained as described (9.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 26415-26420Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Dephosphorylation was initiated by addition of inverted membrane vesicles (1 mg/ml) containing KdpD derivatives and, when indicated, 20 mm MgCl2 and 20 μm ATPγS. At different times, aliquots were removed, and the reaction was stopped by addition of SDS sample buffer (26.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206615) Google Scholar). All samples were immediately subjected to SDS-PAGE. Shortly before stopping SDS-PAGE, an [γ-32P]ATP standard was loaded on the gels. Gels were dried, and phosphorylation of KdpE was detected and quantified as described above. Protein was assayed by a modified Lowry method (27.Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7113) Google Scholar), using bovine serum albumin as a standard. Immunodetection of KdpD, KdpFABC, and His-tagged proteins was performed with polyclonal antibodies against KdpD and KdpFABC, respectively, and monoclonal penta-His antibodies according to a previous study (4.Voelkner P. Puppe W. Altendorf K. Eur. J. Biochem. 1993; 217: 1019-1026Crossref PubMed Scopus (60) Google Scholar). A short version of KdpD comprising the N-terminal domain (amino acids 1–395) followed by six His residues (KdpD/1–395(6His)) was generated by insertion of six codons for His and three stop codons at the appropriate site in kdpD. This gene was overexpressed in E. coli strain TKR2000, which was transformed with plasmid pPV5-3/1–395(6His) (data not shown). Localization studies indicated that KdpD/1–395(6His) was membrane-associated (Fig. 3, lane 2); none of the protein was found in the cytoplasm (Fig. 3, lane 3). KdpD/1–395(6His) could either be completely solubilized by detergent (data not shown) or partially detached from the membrane under low ionic conditions (Fig.3, lanes 4 and 5) (see also “Experimental Procedures”). Protein obtained by both methods was used for purification. As illustrated in Fig. 4, KdpD/1–395(6His) binds either in the presence or absence of detergent specifically to Ni-NTA agarose and can be eluted from the resin by increasing the imidazole concentration to 100 mm. Because under low ionic buffer conditions only membrane-associated proteins are released, purification of KdpD/1–395(6His) reaches about 95% homogeneity, whereas in the presence of detergent two major contaminating proteins are detectable (Fig. 4).Figure 4Purification of KdpD/1–395(6His) by Ni-NTA affinity chromatography. Purification procedure was carried out as described under “Experimental Procedures.” Shown is a Coomassie-stained SDS gel. Lane 1, LDAO- solubilized proteins (20 μg); lane 2, unbound protein (flow-through) (20 μg); lane 3, wash fraction (equal volume as inlanes 1 and 2); lane 4, with imidazole-eluted protein in detergent (3 μg); lane 5, with imidazole-eluted protein after protein was released from the membrane under low ionic buffer conditions without detergent (3 μg).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The N-terminal domain is highly conserved among all thus far known KdpD sequences of various microorganisms. Moreover, it contains a highly conserved region, which is very similar to a classical ATP-binding site (Walker A and Walker B motifs) (Fig. 2). Direct biochemical evidence for binding of ATP to this site was obtained by photoaffinity labeling with 8-azido-[α-32P]ATP. The purified and reconstituted full length sensor kinase, carrying a thrombin cleavage site at amino acid position 395 (KdpD/Th(6His)) to separate the ATP-binding sites of the N-terminal and C-terminal domain, was first labeled with 8-azido-[α-32P]ATP and then treated with thrombin. The protein fragments were loaded on an SDS gel, and the corresponding autoradiography was performed (see Fig.5). Besides the labeled and uncleaved KdpD, two labeled fragments were detectable; however, their size did not correspond to the expected fragment size after thrombin cleavage. Determination of the N-terminal sequences of these fragments confirmed that the large (∼29 kDa) fragment represents amino acids 1–240 and thus compromises the ATP-binding site within the N-terminal domain of KdpD. The amount of protein of the large (∼35-kDa) fragment was too low to determine the N-terminal sequence, thus the corresponding amino acid composition of this fragment is unknown. The same experiment was performed after preincubation of the protein with an excess of nonradioactive ATP. As shown in Fig. 5 (lane 3), labeled full length KdpD but no labeled fragments were detectable indicating the specificity for binding of ATP. Labeling of KdpD, in which the ATP-binding site was inactivated (KdpD/G37A, K38A, T39C) was detectable, although the value reached only 10% compared with that of wild-type KdpD (data not shown). As shown in Fig. 5 (lane 4), the isolated N-terminal domain retains the ability to bind 8-azido-ATP when it is membrane-associated. In contrast, no labeling of the solubilized N-terminal domain was observed, neither after solubilization by detergent nor after treatment with low-ionic buffer (lane 5). The importance of the N-terminal domain for the function of the sensor kinase was shown by complementation experiments as described under “Experimental Procedures.” For this purpose, anEscherichia 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 KdpD/Δ12–395 was used.kdpFABC expression was monitored by Western blotting using antibodies directed against the transport complex, KdpFABC. Using a polyclonal antiserum against KdpFABC, only subunit KdpB is easily detectable (Fig. 6). In confirmation of earlier data, which were based on a transcriptional fusion of thekdpFABC promotor and lacZ (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), it is found that KdpFABC production is drastically reduced in this strain in comparison to a control strain (Fig. 6, lanes 1 and 2). However, transformation of E. coli TKW22692 with plasmid pBD/1–395, encoding the N-terminal domain of KdpD, KdpD/1–395, gave rise to a significant increase in the amount of produced KdpFABC complex (Fig. 6, lane 3). The same effect was detectable for the His-tagged N-terminal domain, KdpD/1–395(6His), indicating that the His-tag does not disturb complementation (data not shown). Remarkably, no increase in KdpFABC production was observed when the ATP-binding site in the N-terminal domain was inactivated by amino acid replacements (Fig. 6, lane 4). These results clearly indicate the importance of the ATP-binding site in the hydrophilic N-terminal domain of KdpD and provide evidence for the first time of an interaction of this domain with the rest of the protein. The sensor kinase KdpD is characterized by two enzymatic activities: kinase and phosphatase activity. In vitro, phosphatase activity is high in the presence of ATP or nonhydrolyzable ATP analogues, whereas in the absence of ATP dephosphorylation of KdpE∼P is very slow. However, KdpD constructs lacking the ATP-binding site of the N-terminal domain (KdpD/Δ12–228 or KdpD/Δ12–395) by truncation 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 Scholar), (Fig.7 A). To test whether the N-terminal domain can complement the truncated KdpD in vitro, plasmids pBD6-92, encoding KdpD/Δ12–395, and pBD3/1–395(6His), encoding the N-terminal domain of KdpD, were cotransformed in E. coli strain TKR2000. Cells were cultivated, the two kdpD derivatives were overexpressed, and KdpD phosphatase activity was determined after preparation of inverted membrane vesicles. As shown in Fig. 7 B, inverted membrane vesicles containing both the truncated KdpD as well as the N-terminal domain are characterized by a KdpE∼P phosphatase activity, which shows a restored dependence on ATP. These data further support the assumption of a direct interaction of the N-terminal domain (KdpD/1–395) with the rest of the protein and stress the importance of the ATP-binding site located in this region. The stimulus that KdpD senses to activate signal transduction, which results in induction of kdpFABC expression, is believed to be a decrease in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic solute (12.Epstein W. Acta Physiol. Scand. Suppl. 1992; 607: 193-199PubMed Google Scholar). However, there is an ongoing debate as to whether KdpD is a turgor sensor or not (13.Malli 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, 16.Sugiura A. Hirokawa K. Nakashima K. Mizuno T. Mol. Microbiol. 1994; 14: 929-938Crossref PubMed Scopus (81) Google Scholar). Despite this, the nature of the primary stimulus for KdpD is still unknown. Although KdpD shares high similarity to transmitter domains of other histidine kinases, it is unusual in regard of the large input domain. This input (sensor) domain comprises an extended N-terminal cytoplasmic region, four transmembrane domains, and part of the C-terminal domain (Fig. 1). Three regions within the input domain have been identified to be important for the activity of KdpD: (i) A cluster of positively charged Arg residues close to transmembrane domain four (TM4) is involved in the shift between 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). (ii) The four transmembrane domains seem to be important for the correct positioning of N- and C-terminal domains to each other as well as for perception and/or 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, 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, 22.Jung K. Heermann R. Meyer M. Altendorf K. Biochim. Biophys. Acta. 1998; 1372: 311-322Crossref PubMed Scopus (19) Google Scholar). (iii) Amino acids 12–128, including a slightly modified Walker A site, seem to be important for the regulation 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 describe the overexpression and purification of the N-terminal domain of KdpD as well as its functional importance. Although the Walker A motif found in the N-terminal domain of KdpD is slightly different from a classical motif (an additional amino acid is found between the first and second Gly), photoaffinity labeling by 8-azido-[α-32P]ATP shown here provides direct evidence that the N-terminal domain binds ATP. This has been shown for the full length sensor kinase and for the separately produced domain. The other known example for the same modification of the Walker A motif is found in the dethiobiotin synthetase (28.Huang W. Jia J. Gibson K.J. Taylor W.S. Rendina A.R. Schneider G. Lindqvist Y. Biochemistry. 1995; 34: 10985-10995Crossref PubMed Scopus (56) Google Scholar), for which ATP binding was shown. ATP binding was detected only for the membrane-associated N-terminal domain, but not for the solubilized peptide. Although the N-terminal domain is mainly composed of hydrophilic amino acid residues, there is a stretch of 17 hydrophobic amino acid residues around 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). From the differences found in the ability to bind azido-ATP, it is concluded that only the membrane-associated domain retains the proper folding to accommodate the nucleotide. It has been shown for the ATP-sensitive channel (K ATP channel), which is blocked by intracellular ATP, that binding affinity changes significantly depending on the membrane phospholipid phosphatidylinositol-4,5-bis-phosphate (29.Shyng S.L. Nichols C.G. Science. 1998; 282: 1138-1141Crossref PubMed Scopus (483) Google Scholar). Because negatively charged phospholipids influence the kinase activity of KdpD (10.Stallkamp I. Dowhan W. Altendorf K. Jung K. Arch. Microbiol. 1999; 172: 295-302Crossref PubMed Scopus (22) Google Scholar), it is conceivable that alterations in the attachment of this domain to the lipid bilayer influences the binding to ATP. The truncated KdpD proteins KdpD/Δ12–395 and KdpD/Δ12–228 but not KdpD/Δ128–391 diminish 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). Interestingly, these are the only known examples of KdpD derivatives, which influence the response to K+ limitation. Although other amino acid replacements within KdpD alter the response to osmolarity and/or to an increase in the external K+ concentration, none of them influence 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 present study, we have shown that separate expression of the N-terminal domain significantly increases kdpFABC expression under K+-limiting growth conditions. This result indicates the importance of this domain for signal transduction and provides evidence for an interaction between the N-terminal domain and the C-terminal transmitter domain of KdpD. It has been described before that truncation of amino acids 12–128 causes deregulation of the phosphatase activity in vitro. In the presence of ATP or nonhydrolyzable ATP analogues, dephosphorylation of KdpE∼P is fast, whereas in the absence of ATP only a very slow rate is observed (18.Jung K. Altendorf K. J. Biol. Chem. 1998; 273: 17406-17410Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Truncated KdpD derivatives, in which amino acids 12–128 are missing, are characterized by an ATP-independent 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 dependence of the dephosphorylation of KdpE∼P by KdpD/Δ12–395 is restored in the presence of the separately produced N-terminal domain. The physiological role of the ATP-binding site within the N-terminal domain is still puzzling. Interestingly, under conditions of an osmotic upshock (30.Ohwada T. Sagisaka S. Arch. Biochem. Biophys. 1987; 259: 157-163Crossref PubMed Scopus (56) Google Scholar) as well as under K+-limiting growth conditions 3K. Jung, unpublished observation. a sudden increase of the intracellular ATP concentration is observed. Because ATP binding affects the phosphatase activity of KdpD, a role of the intracellular ATP concentration as a primary stimulus for KdpD to regulate the ratio between kinase and phosphatase activities is conceivable. However, the direct link between the increased intracellular ATP concentration and binding of ATP to the N-terminal domain is unproved. In summary, the results presented here indicate that the N-terminal domain is essential for modulating the phosphatase activity of KdpD and thus proper signal transduction. The data provide direct evidence for the existence of an ATP-binding site within this domain. The role of this binding site remains unclear. KdpD activity can be restoredin vivo as well as in vitro from two separately produced domains providing indirect evidence for the interaction between the N-terminal domain and the rest of the protein. Finally, the purification protocol for the N-terminal domain presented here provides the prerequisite to characterize this interaction in more detail. We thank Dr. Roland Schmid for N-terminal determinations of KdpD proteolysis fragments and Mechthild Krabusch for technical assistance. We also thank Dr. Wolf Epstein, University of Chicago, for critical reading of the manuscript." @default.
• W1990391674 created "2016-06-24" @default.
• W1990391674 creator A5025003953 @default.
• W1990391674 creator A5042759458 @default.
• W1990391674 creator A5065465937 @default.
• W1990391674 date "2000-06-01" @default.
• W1990391674 modified "2023-10-06" @default.
• W1990391674 title "The Hydrophilic N-terminal Domain Complements the Membrane-anchored C-terminal Domain of the Sensor Kinase KdpD ofEscherichia coli" @default.
• W1990391674 cites W124504601 @default.
• W1990391674 cites W1509597125 @default.
• W1990391674 cites W1537831154 @default.
• W1990391674 cites W1567209750 @default.
• W1990391674 cites W1792353848 @default.
• W1990391674 cites W1802673126 @default.
• W1990391674 cites W1963541451 @default.
• W1990391674 cites W1965209959 @default.
• W1990391674 cites W1993918760 @default.
• W1990391674 cites W1995249391 @default.
• W1990391674 cites W1997991989 @default.
• W1990391674 cites W2009828781 @default.
• W1990391674 cites W2028622989 @default.
• W1990391674 cites W2028883837 @default.
• W1990391674 cites W2033313498 @default.
• W1990391674 cites W2036111399 @default.
• W1990391674 cites W2054212257 @default.
• W1990391674 cites W2058763205 @default.
• W1990391674 cites W2060913542 @default.
• W1990391674 cites W2074001058 @default.
• W1990391674 cites W2084991434 @default.
• W1990391674 cites W2100837269 @default.
• W1990391674 cites W2102131116 @default.
• W1990391674 cites W2109109270 @default.
• W1990391674 cites W2117474658 @default.
• W1990391674 cites W2160084442 @default.
• W1990391674 cites W2162641149 @default.
• W1990391674 cites W2163864322 @default.
• W1990391674 cites W4295127969 @default.
• W1990391674 doi "https://doi.org/10.1074/jbc.m000093200" @default.
• W1990391674 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10747873" @default.
• W1990391674 hasPublicationYear "2000" @default.
• W1990391674 type Work @default.
• W1990391674 sameAs 1990391674 @default.
• W1990391674 citedByCount "34" @default.
• W1990391674 countsByYear W19903916742013 @default.
• W1990391674 countsByYear W19903916742014 @default.
• W1990391674 countsByYear W19903916742017 @default.
• W1990391674 countsByYear W19903916742020 @default.
• W1990391674 countsByYear W19903916742022 @default.
• W1990391674 crossrefType "journal-article" @default.
• W1990391674 hasAuthorship W1990391674A5025003953 @default.
• W1990391674 hasAuthorship W1990391674A5042759458 @default.
• W1990391674 hasAuthorship W1990391674A5065465937 @default.
• W1990391674 hasBestOaLocation W19903916741 @default.
• W1990391674 hasConcept C104317684 @default.
• W1990391674 hasConcept C12554922 @default.
• W1990391674 hasConcept C134306372 @default.
• W1990391674 hasConcept C143065580 @default.
• W1990391674 hasConcept C185592680 @default.
• W1990391674 hasConcept C2779664074 @default.
• W1990391674 hasConcept C31258907 @default.
• W1990391674 hasConcept C32619005 @default.
• W1990391674 hasConcept C33923547 @default.
• W1990391674 hasConcept C36503486 @default.
• W1990391674 hasConcept C41008148 @default.
• W1990391674 hasConcept C41625074 @default.
• W1990391674 hasConcept C515207424 @default.
• W1990391674 hasConcept C55493867 @default.
• W1990391674 hasConcept C57711820 @default.
• W1990391674 hasConcept C86803240 @default.
• W1990391674 hasConcept C95444343 @default.
• W1990391674 hasConceptScore W1990391674C104317684 @default.
• W1990391674 hasConceptScore W1990391674C12554922 @default.
• W1990391674 hasConceptScore W1990391674C134306372 @default.
• W1990391674 hasConceptScore W1990391674C143065580 @default.
• W1990391674 hasConceptScore W1990391674C185592680 @default.
• W1990391674 hasConceptScore W1990391674C2779664074 @default.
• W1990391674 hasConceptScore W1990391674C31258907 @default.
• W1990391674 hasConceptScore W1990391674C32619005 @default.
• W1990391674 hasConceptScore W1990391674C33923547 @default.
• W1990391674 hasConceptScore W1990391674C36503486 @default.
• W1990391674 hasConceptScore W1990391674C41008148 @default.
• W1990391674 hasConceptScore W1990391674C41625074 @default.
• W1990391674 hasConceptScore W1990391674C515207424 @default.
• W1990391674 hasConceptScore W1990391674C55493867 @default.
• W1990391674 hasConceptScore W1990391674C57711820 @default.
• W1990391674 hasConceptScore W1990391674C86803240 @default.
• W1990391674 hasConceptScore W1990391674C95444343 @default.
• W1990391674 hasIssue "22" @default.
• W1990391674 hasLocation W19903916741 @default.
• W1990391674 hasOpenAccess W1990391674 @default.
• W1990391674 hasPrimaryLocation W19903916741 @default.
• W1990391674 hasRelatedWork W1993740563 @default.
• W1990391674 hasRelatedWork W2027376329 @default.
• W1990391674 hasRelatedWork W2032161274 @default.
• W1990391674 hasRelatedWork W2064011811 @default.
• W1990391674 hasRelatedWork W2782497155 @default.
• W1990391674 hasRelatedWork W2791663709 @default.
• W1990391674 hasRelatedWork W2952778583 @default.

• next