Crystal structures, metal activation, and DNA-binding properties of two-domain IdeR from Mycobacterium tuberculosis.
The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.
Wisedchaisri
Goragot
G
Department of Biochemistry and Biomolecular Structure Center, University of Washington, Seattle, Washington 98195, USA.
Chou
C James
CJ
Wu
Meiting
M
Roach
Claudia
C
Rice
Adrian E
AE
Holmes
Randall K
RK
Beeson
Craig
C
Hol
Wim G J
WG
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D046911:Macromolecular Substances; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C096348:IdeR protein, Mycobacterium tuberculosis; D046911:Macromolecular Substances; D008670:Metals; D011994:Recombinant Proteins; D012097:Repressor Proteins