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

Wisedchaisri, Goragot and Chou, C. James and Wu, Meiting and Roach, Claudia and Rice, Adrian E. and Holmes, Randall K. and Beeson, Craig and Hol, Wim G. J. (2007) Crystal Structures, Metal Activation, and DNA-Binding Properties of Two-Domain IdeR from Mycobacterium tuberculosis. Biochemistry, 46 (2). pp. 436-447. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170131-140943217

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Abstract

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 Co^(2+) and Ni^(2+) 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 Ni^(2+) concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co^(2+) required for activation remains the same. We have determined the crystal structures of Ni^(2+)-activated 2D-IdeR at 1.96 Å resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 Å and 2.6 Å, 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 His_6. This C-terminal tail promotes crystal contacts by forming a β-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.


Item Type:Article
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http://dx.doi.org/10.1021/bi0609826DOIArticle
http://pubs.acs.org/doi/full/10.1021/bi0609826PublisherArticle
http://pubs.acs.org/doi/suppl/10.1021/bi0609826Related ItemSupporting Information
Additional Information:© 2007 American Chemical Society. Received 17 May 2006. Published online 19 December 2006. Published in print 1 January 2007. We gratefully acknowledge the use of the Advanced Photon Source (APS) beamline 19-ID (SBC-CAT) at Argonne National Laboratory and the Advanced Light Source (ALS) beamline 8.2.2 (HHMI) at Lawrence Berkeley National Laboratory and their staffs for technical assistance. Use of the APS and the ALS is supported by the U.S. Department of Energy.
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Department of Energy (DOE)UNSPECIFIED
Issue or Number:2
Record Number:CaltechAUTHORS:20170131-140943217
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170131-140943217
Official Citation:Crystal Structures, Metal Activation, and DNA-Binding Properties of Two-Domain IdeR from Mycobacterium tuberculosis Goragot Wisedchaisri, C. James Chou, Meiting Wu, Claudia Roach, Adrian E. Rice, Randall K. Holmes, Craig Beeson, and Wim G. J. Hol Biochemistry 2007 46 (2), 436-447 DOI: 10.1021/bi0609826
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73898
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:01 Feb 2017 00:35
Last Modified:03 Oct 2019 16:32

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