Bacterial sensor kinase TodS interacts with agonistic and antagonistic signals.
The TodS/TodT two-component system controls expression of the toluene dioxygenase (TOD) pathway for the metabolism of toluene in Pseudomonas putida DOT-T1E. TodS is a sensor kinase that ultimately controls tod gene expression through its cognate response regulator, TodT. We used isothermal titration calorimetry to study the binding of different compounds to TodS and related these findings to their capacity to induce gene expression in vivo. Agonistic compounds bound to TodS and induced gene expression in vivo. Toluene was a powerful agonist, but ortho-substitutions of toluene reduced or abolished in vivo responses, although TodS recognized o-xylene with high affinity. These compounds were called antagonists. We show that agonists and antagonists compete for binding to TodS both in vitro and in vivo. The failure of antagonists to induce gene expression in vivo correlated with their inability to stimulate TodS autophosphorylation in vitro. We propose intramolecular TodS signal transmission, not molecular recognition of compounds by TodS, to be the phenomenon that determines whether a given compound will lead to activation of expression of the tod genes. Molecular modeling identified residues F46, I74, F79, and I114 as being potentially involved in the binding of effector molecules. Alanine substitution mutants of these residues reduced affinities (2- to 345-fold) for both agonistic and antagonistic compounds. Our data indicate that determining the inhibitory activity of antagonists is a potentially fruitful alternative to design specific two-component system inhibitors for the development of new drugs to inhibit processes regulated by two-component systems.
Busch
Andreas
A
Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, C/Profesor Albareda, 1, 18008 Granada, Spain.
Lacal
Jesús
J
Martos
Ariadna
A
Ramos
Juan L
JL
Krell
Tino
T
D001426:Bacterial Proteins / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D005786:Gene Expression Regulation; D006838:Hydrocarbons / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry; D014050:Toluene / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects
D001426:Bacterial Proteins; D006838:Hydrocarbons; D008024:Ligands; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D014050:Toluene; D011494:Protein Kinases
Annu Rev Biochem. 2000;69:183-215
10966457
J Mol Biol. 2000 Aug 11;301(2):415-31
10926518
Mol Biol Evol. 2000 Dec;17(12):1956-70
11110912
Nucleic Acids Res. 2001 Jan 1;29(1):55-7
11125048
Microbes Infect. 2001 Apr;3(5):417-24
11369279
J Biol Chem. 2001 Aug 17;276(33):30827-33
11404360
Proteins. 2001;Suppl 5:39-46
11835480
Bioorg Med Chem. 2002 Apr;10(4):855-67
11836091
Chem Biol. 2002 Nov;9(11):1209-17
12445771
J Bacteriol. 2002 Dec;184(24):7062-7
12446657
Nat Struct Biol. 2002 Dec;9(12):912-7
12434151
J Biol Chem. 2003 May 16;278(20):18434-9
12639952
Biochemistry. 2003 May 20;42(19):5917-24
12741850
J Bacteriol. 2003 Aug;185(16):4755-63
12896994
J Bacteriol. 2003 Aug;185(16):4956-72
12897016
Mol Microbiol. 2003 Oct;50(1):219-30
14507376
J Biol Chem. 2003 Oct 3;278(40):39189-96
12867417
FEMS Microbiol Lett. 2004 Feb 16;231(2):277-81
14987775
Curr Med Chem. 2004 Mar;11(6):765-73
15032730
J Biol Chem. 1989 Sep 5;264(25):14940-6
2670929
J Mol Biol. 1992 May 5;225(1):93-105
1583696
Cell. 1996 Jan 12;84(1):165-74
8548821
Appl Environ Microbiol. 1997 Feb;63(2):652-60
9023943
Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1453-8
9037074
J Bacteriol. 1998 Mar;180(5):1063-71
9495743
Gene. 1999 May 17;232(1):69-76
10333523
J Bacteriol. 1999 Sep;181(17):5309-16
10464202
Trends Microbiol. 2005 Feb;13(2):52-6
15680762
J Biol Chem. 2005 May 27;280(21):20887-93
15767250
Cell. 2005 Aug 12;122(3):461-72
16096064
Nucleic Acids Res. 2006 Jan 1;34(Database issue):D247-51
16381856
J Mol Biol. 2006 Mar 10;356(5):1193-206
16406409
Microb Ecol. 2006 Feb;51(2):166-76
16435167
Proc Natl Acad Sci U S A. 2006 May 23;103(21):8191-6
16702539
J Bacteriol. 2006 Jun;188(12):4169-82
16740923
Nat Rev Microbiol. 2006 Sep;4(9):705-9
16894339
Microbiol Mol Biol Rev. 2006 Dec;70(4):910-38
17158704
J Biol Chem. 2007 Jun 1;282(22):16308-16
17416591
J Biol Chem. 2000 Dec 8;275(49):38900-4
10978341