Domain structure of virulence-associated response regulator PhoP of Mycobacterium tuberculosis: role of the linker region in regulator-promoter interaction(s).
The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met(1) to Arg(138) of PhoP constitute the minimal functional PhoPN, we identified Arg(150) as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.
Pathak
Anuj
A
Institute of Microbial Technology, Council of Scientific and Industrial Research, Sector 39A, Chandigarh 160036, India.
Goyal
Rajni
R
Sinha
Akesh
A
Sarkar
Dibyendu
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008055:Lipids / Q000096:biosynthesis / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008055:Lipids; D037521:Virulence Factors; C061064:PhoP protein, Bacteria
J Exp Med. 2004 Mar 1;199(5):649-59
14981115
Microbiology (Reading). 2005 Dec;151(Pt 12):3979-3987
16339942
Cell Microbiol. 2004 Mar;6(3):277-87
14764111
Mol Microbiol. 2001 Jul;41(1):179-87
11454210
FEBS Lett. 2006 Oct 2;580(22):5328-38
16979633
Trends Microbiol. 2008 Nov;16(11):528-34
18835713
Biochemistry. 2007 Dec 25;46(51):14751-61
18052041
Proc Natl Acad Sci U S A. 1999 Oct 12;96(21):11792-7
10518529
Methods Enzymol. 1985;115:420-30
4079796
J Bacteriol. 2009 Dec;191(24):7466-76
19820095
J Bacteriol. 2000 Dec;182(23):6592-7
11073900
Structure. 2002 May;10(5):701-13
12015152
Cell Host Microbe. 2008 Feb 14;3(2):97-103
18312844
J Mol Biol. 1998 Sep 4;281(5):857-70
9719640
J Bacteriol. 2008 Feb;190(4):1317-28
18065544
PLoS Pathog. 2008 Feb 8;4(2):e33
18282096
Nature. 1999 Nov 4;402(6757):79-83
10573420
J Bacteriol. 2004 Feb;186(4):1182-90
14762014
J Biol Chem. 2006 Apr 7;281(14):9659-66
16434396
J Bacteriol. 2003 Jul;185(14):4186-94
12837793
Trends Biochem Sci. 2007 May;32(5):225-34
17433693
J Bacteriol. 2008 Nov;190(21):7068-78
18757548
Infect Immun. 2003 Aug;71(8):4684-90
12874349
Structure. 2002 Feb;10(2):153-64
11839301
Tuberculosis (Edinb). 2007 Mar;87(2):78-86
17030019
Mol Microbiol. 1999 Oct;34(2):257-67
10564470
Gene. 1989 Apr 15;77(1):51-9
2744487
J Bacteriol. 2008 Feb;190(4):1329-34
18065542
Proc Natl Acad Sci U S A. 2003 May 13;100(10):6121-6
12724526
Trends Microbiol. 2004 Nov;12(11):500-8
15488391
J Bacteriol. 2004 Mar;186(5):1493-502
14973033
Proc Natl Acad Sci U S A. 1959 Dec;45(12):1801-4
16590578
Structure. 1997 Jan 15;5(1):109-24
9016718
J Bacteriol. 2001 Apr;183(7):2204-11
11244058
Curr Opin Microbiol. 2002 Apr;5(2):135-41
11934608
Eur J Biochem. 1988 Mar 15;172(3):579-84
3127210
J Bacteriol. 2003 Jan;185(1):317-24
12486069
J Biochem. 2001 Mar;129(3):343-50
11226872
J Biol Chem. 2006 Jan 20;281(3):1313-6
16326699
J Biol Chem. 1956 Jun;220(2):893-904
13331947
Cell Microbiol. 2003 Jun;5(6):405-15
12780778
Mol Microbiol. 1996 Jun;20(6):1155-63
8809768
J Biol Chem. 2004 May 14;279(20):21257-65
15001577
Microbes Infect. 2006 Jan;8(1):183-90
16182591
J Bacteriol. 2004 Apr;186(8):2476-80
15060051
Mol Microbiol. 2006 Apr;60(2):312-30
16573683
Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11554-9
10500215
J Mol Biol. 2010 May 21;398(5):647-56
20363229