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J. Biol. Chem., Vol. 280, Issue 41, 34776-34785, October 14, 2005

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Reassessment of Protein Stability, DNA Binding, and Protection of Mycobacterium smegmatis Dps^*

From the National Research Council Institute of Molecular Biology and Pathology, Department of Biochemical Sciences "A. Rossi-Fanelli," University of Rome "La Sapienza," 00185 Rome, Italy

The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji (Gupta, S., and Chatterji, D. (2003) J. Biol. Chem. 278, 5235-5241), in which the C terminus that is used for binding DNA contains a histidine tag, have been characterized in parallel. The native dodecamer dissociated reversibly into dimers above pH 7.5 and below pH 6.0, with apparent pK_a values of 7.65 and 4.75; at pH 4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu¹⁵⁷ and Arg⁹⁹ located at the 3-fold symmetry axes and to protonation of Asp⁶⁶ hydrogen-bonded to Lys³⁶ across the dimer interface, respectively. The C-terminal tag did not affect subunit dissociation, but altered DNA binding dramatically. At neutral pH, protonation of the histidine tag promoted DNA condensation, whereas in the native C terminus, compensation of negative and positive charges led to DNA binding without condensation. This different mode of interaction with DNA has important functional consequences as indicated by the failure of the native protein to protect DNA from DNase-mediated cleavage and by the efficiency of the tagged protein in doing so as a result of DNA sequestration in the condensates. Chemical protection of DNA from oxidative damage is realized by Dps proteins in a multistep iron oxidation/uptake/mineralization process. Dimers have a decreased protection efficiency due to disruption of the dodecamer internal cavity, where iron is deposited and mineralized after oxidation at the ferroxidase center.

Received for publication, March 2, 2005 , and in revised form, June 24, 2005.

The atomic coordinates and structure factors (code 1UVH) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Ministero Istruzione Università e Ricerca "Biologia Strutturale e Dinamica di Proteine Redox," the Centro di Eccellenza Biologia e Medicina Molecolare, and Fondo Investiment Ricerca di Base 2001 (to E. C.). 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 To whom correspondence should be addressed: Istituto di Biologia e Patologia Molecolari CNR, Dipartimento di Scienze Biochimiche, Università di Roma "La Sapienza," P. le A. Moro, 5, 00185 Roma, Italy. Tel.: 39-6-494-0543/39-6-4991-0761; Fax: 39-6-444-0062; E-mail: emilia.chiancone{at}uniroma1.it.

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