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J. Biol. Chem., Vol. 283, Issue 25, 17681-17690, June 20, 2008

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Molecular Features Governing the Stability and Specificity of Functional Complex Formation by Mycobacterium tuberculosis CFP-10/ESAT-6 Family Proteins^*

Kirsty L. Lightbody, Dariush Ilghari¹, Lorna C. Waters, Gemma Carey, Mark A. Bailey, Richard A. Williamson^¶, Philip S. Renshaw, and Mark D. Carr²

From the Department of Biochemistry, Henry Wellcome Building, University of Leicester, Leicester, LE1 9HN, United Kingdom, the Iran Ministry of Health and Medical Education, MOHME Building, Sharake Qods, Tehran, Iran 1467664961, and the ^¶Department of Biosciences, University of Kent, Canterbury, Kent, CT2 7NJ, United Kingdom

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287·Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10·ESAT-6. This approach demonstrated that neither Rv0287·Rv0288 nor the CFP-10·ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287·Rv0288 and CFP-10·ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10·ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Received for publication, January 7, 2008 , and in revised form, March 20, 2008.

^* This work was supported in part by Wellcome Trust Value in People Award 0786607/Z/05/Z (to P. R.) and Wellcome Trust Project Grant 080085/Z/06/Z. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a Ph.D. student grant awarded by the Iran Ministry of Health and Medical Education.

² To whom correspondence should be addressed. Tel.: 44-116-229-7075; Fax: 44-116-229-7018; E-mail: mdc12{at}le.ac.uk.

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