title: Ligand-induced folding of a two-component signaling receiver domain.
abstract: To survive and adapt to environmental changes, bacteria commonly use two-component signaling systems. Minimally, these pathways use histidine kinases (HKs) to detect environmental signals, harnessing these to control phosphorylation levels of receiver (REC) domains of downstream response regulators that convert this signal into physiological responses. Studies of several prototypical REC domains suggest that phosphorylation shifts these proteins between inactive and active structures that are globally similar and well-folded. However, it is unclear how globally these findings hold within REC domains in general, particularly when they are considered within full-length proteins. Here, we present EL_LovR, a full-length REC-only protein that is phosphorylated in response to blue light in the marine α-proteobacterium, Erythrobacter litoralis HTCC2594. Notably, EL_LovR is similar to comparable REC-only proteins used in bacterial general stress responses, where genetic evidence suggests that their potent phosphatase activity is important to shut off such systems. Size exclusion chromatography, light scattering, and solution NMR experiments show that EL_LovR is monomeric and unfolded in solution under conditions routinely used for other REC structure determinations. Addition of Mg(2+) and phosphorylation induce progressively greater degrees of tertiary structure stabilization, with the solution structure of the fully activated EL_LovR adopting the canonical receiver domain fold. Parallel functional assays show that EL_LovR has a fast dephosphorylation rate, consistent with its proposed function as a phosphate sink that depletes the HK phosphoryl group, promoting the phosphatase activity of this enzyme. Our findings demonstrate that EL_LovR undergoes substantial ligand-dependent conformational changes that have not been reported for other RRs, expanding the scope of conformational changes and regulation used by REC domains, critical components of bacterial signaling systems.
authors: Departments of Biophysics and Biochemistry, UT Southwestern Medical Center , Dallas, Texas 75390-8816, United States.
authors: Victor J
authors: VJ
authors: Ocasio
authors: Fernando
authors: F
authors: Corrêa
authors: Kevin H
authors: KH
authors: Gardner
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D017510:Protein Folding; D015398:Signal Transduction; D042301:Sphingomonadaceae / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008024:Ligands
references: Proc Natl Acad Sci U S A. 2001 Mar 27;98(7):4095-100
references: 11274434
references: Biochemistry. 1995 Jan 31;34(4):1413-24
references: 7827089
references: Curr Opin Microbiol. 2010 Apr;13(2):142-9
references: 20211578
references: Biochemistry. 1993 Dec 14;32(49):13375-80
references: 8257674
references: J Bacteriol. 2006 Nov;188(21):7328-30
references: 17050920
references: J Mol Biol. 1994 May 13;238(4):489-95
references: 8176739
references: Biochemistry. 2012 Nov 13;51(45):9067-75
references: 23102260
references: J Mol Biol. 1999 Jan 15;285(2):675-87
references: 9878437
references: J Phys Chem B. 2014 Apr 17;118(15):4088-97
references: 24673507
references: J Biomol NMR. 1995 Nov;6(3):277-93
references: 8520220
references: Nat Struct Biol. 2001 Jan;8(1):52-6
references: 11135671
references: Mol Microbiol. 2010 Oct;78(2):290-304
references: 20735776
references: Protein Expr Purif. 1999 Feb;15(1):34-9
references: 10024467
references: Annu Rev Microbiol. 2009;63:133-54
references: 19575571
references: J Mol Biol. 2000 Mar 31;297(3):543-51
references: 10731410
references: Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W500-2
references: 15215436
references: Biochemistry. 1990 Jun 12;29(23):5436-42
references: 2201404
references: J Biol Chem. 2001 May 11;276(19):16425-31
references: 11279165
references: J Bacteriol. 2007 Aug;189(16):5987-95
references: 17545283
references: J Bacteriol. 1998 Jul;180(14):3563-9
references: 9657998
references: J Bacteriol. 2012 Jun;194(12):3038-49
references: 22408156
references: J Am Chem Soc. 2005 Nov 2;127(43):14970-1
references: 16248604
references: Curr Opin Microbiol. 2010 Apr;13(2):113-5
references: 20219418
references: Annu Rev Biochem. 2000;69:183-215
references: 10966457
references: J Bacteriol. 2003 Nov;185(21):6385-91
references: 14563873
references: Structure. 2012 Aug 8;20(8):1363-73
references: 22727815
references: Biochemistry. 1999 Dec 14;38(50):16424-31
references: 10600103
references: J Biomol NMR. 1994 Sep;4(5):603-14
references: 22911360
references: Proc Natl Acad Sci U S A. 1999 Dec 21;96(26):14789-94
references: 10611291
references: J Mol Biol. 2013 Jul 10;425(13):2372-81
references: 23648838
references: Nature. 1999 Dec 23-30;402(6764):894-8
references: 10622255
references: Nature. 1999 Jul 15;400(6741):289-93
references: 10421374
references: J Mol Biol. 2004 Dec 3;344(4):919-28
references: 15544802
references: J Biol Chem. 1991 Aug 15;266(23):15511-9
references: 1869568
references: Science. 1997 Mar 14;275(5306):1658-61
references: 9054362
references: Proteins. 1993 Sep;17(1):75-86
references: 8234246
references: Biochemistry. 2014 Jan 21;53(2):311-22
references: 24364624
references: Cell. 2009 Dec 11;139(6):1109-18
references: 20005804
references: Curr Opin Microbiol. 2010 Apr;13(2):160-7
references: 20080056
references: Curr Opin Microbiol. 2013 Apr;16(2):156-62
references: 23352354
references: Biochemistry. 2013 Jul 9;52(27):4656-66
references: 23806044
references: Proc Natl Acad Sci U S A. 2007 Nov 13;104(46):18241-6
references: 17986614
references: J Mol Biol. 1998 May 8;278(3):507-14
references: 9600834
references: Curr Opin Microbiol. 2009 Apr;12(2):152-60
references: 19246239
references: Trends Biochem Sci. 2007 May;32(5):225-34
references: 17433693
references: Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):13160-5
references: 12237413
references: Curr Opin Microbiol. 2010 Apr;13(2):177-83
references: 20133180
references: Annu Rev Microbiol. 2012;66:325-47
references: 22746333
references: Structure. 1999 Dec 15;7(12):1505-15
references: 10647181
references: Science. 2001 Mar 23;291(5512):2429-33
references: 11264542
references: Anal Biochem. 1976 May 7;72:248-54
references: 942051
references: J Biol Chem. 1991 May 5;266(13):8348-54
references: 1902474
references: Biomol NMR Assign. 2011 Apr;5(1):55-8
references: 20936511
references: J Mol Biol. 2003 Aug 1;331(1):245-54
references: 12875849
references: Bioinformatics. 2007 Feb 1;23(3):381-2
references: 17121777
references: J Biomol NMR. 2013 Jul;56(3):227-41
references: 23728592
references: J Bacteriol. 2009 Apr;191(7):2419-20
references: 19168610
references: Biochemistry. 1997 Aug 19;36(33):10015-25
references: 9254596
references: J Biol Chem. 2013 Jul 26;288(30):21755-69
references: 23760278
references: J Mol Biol. 2014 Apr 3;426(7):1554-67
references: 24406745
references: Proc Natl Acad Sci U S A. 2014 Dec 16;111(50):17839-44
references: 25468971
references: Mol Microbiol. 2008 Jul;69(2):453-65
references: 18557815
references: Biochemistry. 1995 May 23;34(20):6784-94
references: 7756310