Crystal Structures, Metal Activation, and DNA-Binding Properties of Two-Domain IdeR from Mycobacterium tuberculosis

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• 作者：Goragot Wisedchaisri ; C. James Chou ; Meiting Wu ; Claudia Roach ; Adrian E. Rice ; Randall K. Holmes ; Craig Beeson ; Wim G. J. Hol
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：January 16, 2007
• 年：2007
• 卷：46
• 期：2
• 页码：436 - 447
• 全文大小：1339K
• 年卷期：v.46,no.2(January 16, 2007)
• ISSN：1520-4995

文摘

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake inMycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domainof 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 constantsfor Co²⁺ and Ni²⁺ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinitiesof 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 metalion. However, the Ni²⁺ concentrations required to activate 2D-IdeR for DNA binding appear to be smallerthan 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 Å resolution and its doubledimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 Å and 2.6 Å, the highestresolutions 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 basesand suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structuresreveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminalHis₆. This C-terminal tail promotes crystal contacts by forming a ges/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-sheet with the corresponding tail ofneighboring 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 otherproteins.