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          Institute: MPI für medizinische Forschung     Collection: Max-Planck-Forschungsgruppe Ionenkanalstruktur (Dean R. Madden)     Display Documents



ID: 23074.0, MPI für medizinische Forschung / Max-Planck-Forschungsgruppe Ionenkanalstruktur (Dean R. Madden)
Identification of amino acid residues influencing ligand binding by the periplasmic citrate receptor domain of the sensor kinase CitA
Translation of Title:Identification of amino acid residues influencing ligand binding by the periplasmic citrate receptor domain of the sensor kinase CitA
Authors:Gerharz, Tanja; Reinelt, Stephan; Kaspar, Sibylle; Scapozza, Leonardo; Bott, Michael
Language:English
Date of Publication (YYYY-MM-DD):2003-05-20
Title of Journal:Biochemistry
Journal Abbrev.:Biochem.
Volume:42
Issue / Number:19
Start Page:5917
End Page:5924
Review Status:Peer-review
Audience:Experts Only
Intended Educational Use:No
Abstract / Description: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.
External Publication Status:published
Document Type:Article
Communicated by:Wulf Kaiser
Affiliations:MPI für medizinische Forschung/Nachwuchsgruppe Ion Channel Structure/
Identifiers:URI:http://pubs.acs.org/cgi-bin/article.cgi/bichaw/200... [Full text]
URI:http://pubs.acs.org/cgi-bin/abstract.cgi/bichaw/20... [Abstract]
URI:http://pubs.acs.org/cgi-bin/article.cgi/bichaw/200... [Fulltext PDF]
DOI:10.1021/bi0340595
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