Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA.
The sensor kinase CitA and the response regulator CitB of Klebsiella pneumoniae form the paradigm of a subfamily of bacterial two-component regulatory systems that are capable of sensing tri- or dicarboxylates in the environment and then induce transporters for the uptake of these compounds. We recently showed that the separated periplasmic domain of CitA, termed CitAP (encompasses residues 45-176 supplemented with an N-terminal methionine residue and a C-terminal hexahistidine tag), is a highly specific citrate receptor with a K(d) of 5.5 microM at pH 7. To identify positively charged residues involved in binding the citrate anion, each of the arginine, lysine, and histidine residues in CitAP was exchanged for alanine, and the resulting 17 muteins were analyzed by isothermal titration calorimetry (ITC). In 12 cases, the K(d) for citrate was identical to that of wild-type CitAP or slightly changed (3.9-17.2 microM). In one case (R98A), the K(d) was 6-fold decreased (0.8 microM), whereas in four cases (R66A, H69A, R107A, and K109A) the K(d) was 38- to >300-fold increased (0.2 to >1 mM). The secondary structure of the latter five proteins in their apo-form as deduced from far-UV circular dichroism (CD) spectra did not differ from the apo-form of wild-type CitAP; however, all of them showed an increased thermostability. Citrate increased the melting point (T(m)) of wild-type CitAP and mutein R98A by 6.2 and 9.5 degrees C, respectively, but had no effect on the T(m) of the four proteins with disturbed binding. Three of the residues important for citrate binding (R66, H69, and R107) are highly conserved in the CitA subfamily of sensor kinases, indicating that they might be involved in ligand binding by many of these sensor kinases.
Institut für Biotechnologie 1, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Tanja
T
Gerharz
Stefan
S
Reinelt
Sibylle
S
Kaspar
Leonardo
L
Scapozza
Michael
M
Bott
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D024361:Amino Acids, Basic / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D019343:Citric Acid / Q000378:metabolism*; D004269:DNA, Bacterial / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D024361:Amino Acids, Basic; D001426:Bacterial Proteins; C107075:CitB protein, Klebsiella pneumoniae; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D008024:Ligands; D014157:Transcription Factors; D019343:Citric Acid; D011494:Protein Kinases; C460095:CitA protein, E coli