Identification of Basic Amino Acid Residues Important for Citrate Binding by the Periplasmic Receptor Domain of the Sensor Kinase CitA
| Autor: | Stefan Reinelt, Leonardo Scapozza, Sibylle Kaspar, Tanja Gerharz, Michael Bott |
|---|---|
| Rok vydání: | 2003 |
| Předmět: |
Circular dichroism
chemistry [Protein Kinases] genetics [Klebsiella pneumoniae] Ligands Biochemistry Protein structure metabolism [Transcription Factors] metabolism [Protein Kinases] Alanine chemistry.chemical_classification chemistry [Transcription Factors] Circular Dichroism Escherichia coli Proteins genetics [Protein Kinases] metabolism [Periplasm] genetics [Transcription Factors] metabolism [Citric Acid] Amino acid dpiB protein E coli Klebsiella pneumoniae CitB protein Klebsiella pneumoniae Periplasm Thermodynamics genetics [Escherichia coli Proteins] genetics [Bacterial Proteins] Protein Binding DNA Bacterial chemistry [Bacterial Proteins] metabolism [Bacterial Proteins] Molecular Sequence Data chemistry [Amino Acids Basic] Biology metabolism [Escherichia coli Proteins] Citric Acid chemistry [Escherichia coli Proteins] Bacterial Proteins genetics [DNA Bacterial] ddc:570 Amino Acid Sequence Binding site metabolism [Klebsiella pneumoniae] Histidine Binding Sites Base Sequence Sequence Homology Amino Acid Amino Acids Basic Isothermal titration calorimetry Periplasmic space Protein Structure Tertiary Kinetics Amino Acid Substitution chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Mutagenesis Site-Directed Protein Kinases Transcription Factors |
| Zdroj: | Biochemistry 42, 5917-5924 (2003). doi:10.1021/bi0340595 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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