Energetics of Sequence-Specific Protein-DNA Association: Conformational Stability of the DNA Binding Domain of Integrase Tn916 and Its Cognate DNA Duplex † Stoyan Milev, ‡ Alemayehu A. Gorfe, ‡ Andrey Karshikoff, § Robert T. Clubb, | Hans Rudolf Bosshard, ‡ and Ilian Jelesarov* ,‡ Institute of Biochemistry, UniVersity of Zu ¨ rich, Winterthurerstrasse 190m Room 44 L42, CH-8057 Zu ¨ rich, Switzerland, Center for Structural Biochemistry, Department of Biosciences, NOVUM, Karolinska Institutet, S-141 57 Huddinge, Sweden, and Department of Chemistry and Biochemistry and UCLA-DOE Laboratory of Structural Biology and Genetics, UniVersity of California, 405 Hilgard AVenue, Los Angeles, California 90095 ReceiVed October 1, 2002; ReVised Manuscript ReceiVed January 16, 2003 ABSTRACT: Sequence-specific DNA recognition by bacterial integrase Tn916 involves structural re- arrangements of both the protein and the DNA duplex. Energetic contributions from changes of conformation, thermal motions and soft vibrational modi of the protein, the DNA, and the complex significantly influence the energetic profile of protein-DNA association. Understanding the energetics of such a complicated system requires not only a detailed calorimetric investigation of the association reaction but also of the components in isolation. Here we report on the conformational stability of the integrase Tn916 DNA binding domain and its cognate 13 base pair target DNA duplex. Using a combination of temperature and denaturant induced unfolding experiments, we find that the 74-residue DNA binding domain is compact and unfolds cooperatively with only small deviation from two-state behavior. Scanning calorimetry reveals an increase of the heat capacity of the native protein attributable to increased thermal fluctuations. From the combined calorimetric and spectroscopic experiments, the parameters of protein unfolding are T m ) 43.8 ( 0.3 °C, ∆H m ) 255 ( 18 kJ mol -1 , ∆S m ) 0.80 ( 0.06 kJ mol -1 , and ∆C p ) 5.0 ( 0.8 kJ K -1 mol -1 . The DNA target duplex displays a thermodynamic signature typical of short oligonucleotide duplexes: significant heat absorption due to end fraying and twisting precedes cooperative unfolding and dissociation. The parameters for DNA unfolding and dissociation are ∆H m ) 335 ( 4 kJ mol -1 and ∆C p ) 2.7 ( 0.9 kJ K -1 mol -1 . The results reported here have been instrumental in interpreting the thermodynamic features of the association reaction of the integrase with its 13 base pair target DNA duplex reported in the accompanying paper [Milev et al. (2003) Biochemistry 42, 3481-3491]. Antibiotic resistance of bacteria is spread by promiscuous conjugative transposons (1). One of the most thoroughly studied representatives of the family is the Tn916 element carrying resistance to tetracycline. Excision of the Tn916 transposon requires the formation of a complicated nucleo- protein complex similar to the λ-phage “intasome” (2). In the synaptic complex, the C-terminal catalytic domain of the transposon-encoded integrase is brought to the cleavage site by the N-terminal DNA binding domain, which binds to repeated sequences within the transposon arm. The solution structure of the N-terminal DNA binding domain complexed to a 13 bp DNA duplex has been solved by NMR spectroscopy (3). Unlike most other major groove binders, the Tn916 integrase recognizes its DNA target by positioning the face of a three-stranded -sheet into the major groove. The conformation of the protein is changed in the complex when compared to its free native form (4). 1 Surprisingly, the protein appears more disordered in the DNA-bound state (3). The DNA target site keeps a B-form conformation in the bound state except that the major groove is widened in the middle of the duplex. In the accompanying paper, we describe the energetics of the association reaction of the DNA binding domain with the 13 bp target DNA duplex (5). The reaction exhibits a large negative heat capacity change, ∆C p , which is itself temperature-dependent and cannot be accounted for by the amount of polar and nonpolar surface buried at the complex interface, as would be the case for a rigid body type of association. A steep increase of the heat capacity of the complex was observed before the thermal transition into the unfolded protein and dissociated DNA strands. This behavior could only be interpreted with the help of the thermodynamic parameters of unfolding of the free protein and the duplex DNA described in the present paper (5). † This work was supported in part by the Swiss National Science Foundation. * Corresponding author. Telephone: +41 1 655 5547. Fax: +41 1 635 6805. E-mail: iljel@bioc.unizh.ch ‡ University of Zu ¨rich. § Karolinska Institutet. | University of California, Los Angeles. 1 To facilitate reading, the N-terminal fragment 2-74 of integrase Tn916 and the 13 bp duplex target DNA are called “protein” and “DNA”, respectively. 3492 Biochemistry 2003, 42, 3492-3502 10.1021/bi026936x CCC: $25.00 © 2003 American Chemical Society Published on Web 03/08/2003