TY  - JOUR
AU  - Gerharz, T.
AU  - Reinelt, S.
AU  - Kaspar, S.
AU  - Scapozza, L.
AU  - Bott, M.
TI  - Identification of basic amino acid residues important for citrate binding by the periplasmic receptor domain of the sensor kinase CitA
JO  - Biochemistry
VL  - 42
SN  - 0006-2960
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - PreJuSER-30967
SP  - 5917 - 5924
PY  - 2003
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Amino Acid Sequence
KW  - Amino Acid Substitution
KW  - Amino Acids, Basic: chemistry
KW  - Bacterial Proteins: chemistry
KW  - Bacterial Proteins: genetics
KW  - Bacterial Proteins: metabolism
KW  - Base Sequence
KW  - Binding Sites
KW  - Circular Dichroism
KW  - Citric Acid: metabolism
KW  - DNA, Bacterial: genetics
KW  - Escherichia coli Proteins: chemistry
KW  - Escherichia coli Proteins: genetics
KW  - Escherichia coli Proteins: metabolism
KW  - Kinetics
KW  - Klebsiella pneumoniae: genetics
KW  - Klebsiella pneumoniae: metabolism
KW  - Ligands
KW  - Molecular Sequence Data
KW  - Mutagenesis, Site-Directed
KW  - Periplasm: metabolism
KW  - Protein Binding
KW  - Protein Kinases: chemistry
KW  - Protein Kinases: genetics
KW  - Protein Kinases: metabolism
KW  - Protein Structure, Tertiary
KW  - Sequence Homology, Amino Acid
KW  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
KW  - Thermodynamics
KW  - Transcription Factors: chemistry
KW  - Transcription Factors: genetics
KW  - Transcription Factors: metabolism
KW  - Amino Acids, Basic (NLM Chemicals)
KW  - Bacterial Proteins (NLM Chemicals)
KW  - CitB protein, Klebsiella pneumoniae (NLM Chemicals)
KW  - DNA, Bacterial (NLM Chemicals)
KW  - Escherichia coli Proteins (NLM Chemicals)
KW  - Ligands (NLM Chemicals)
KW  - Transcription Factors (NLM Chemicals)
KW  - Citric Acid (NLM Chemicals)
KW  - Protein Kinases (NLM Chemicals)
KW  - dpiB protein, E coli (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:12741850
UR  - <Go to ISI:>//WOS:000182934300041
DO  - DOI:10.1021/bi0340595
UR  - https://juser.fz-juelich.de/record/30967
ER  -