Protein-Directed DNA Structure. I. Raman Spectroscopy of a High-Mobility-Group Box with Application to Human Sex Reversal † James M. Benevides, ‡ Ging Chan, § Xiang-Jun Lu, | Wilma K. Olson, | Michael A. Weiss,* ,§,⊥ and George J. Thomas, Jr* ,‡ DiVision of Cell Biology and Biophysics, School of Biological Sciences, UniVersity of MissourisKansas City, Kansas City, Missouri 64110, Center for Molecular Oncology, Department of Biochemistry and Molecular Biology and Department of Chemistry, UniVersity of Chicago, Chicago, IL 60637, and Department of Chemistry, Rutgers UniVersity, New Brunswick, NJ 08903 ReceiVed January 8, 1999; ReVised Manuscript ReceiVed October 29, 1999 ABSTRACT: Protein-directed reorganization of DNA underlies mechanisms of transcription, replication, and recombination. A molecular model for DNA reorganization in the regulation of gene expression is provided by the sequence-specific high-mobility-group (HMG) box. Structures of HMG-box complexes with DNA are characterized by expansion of the minor groove, sharp bending toward the major groove, and local unwinding of the double helix. The Raman vibrational signature of such DNA reorganization has been identified in a study of the SRY HMG box, encoded by the human male-determining region of the Y chromosome. We observe in the human SRY-HMG:DNA complex extraordinarily large perturbations to Raman bands associated with vibrational modes of the DNA backbone and accompanying large increases in intensities of Raman bands attributable to base unstacking. In contrast, DNA major- groove binding, as occurs for the bZIP protein GCN4 [Benevides, J. M., Li, T., Lu, X.-J., Srinivasan, A. R., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 548-556], perturbs the Raman signature of DNA only marginally. Raman markers of minor-groove recognition in the human SRY-HMG:DNA complex are due primarily to perturbation of specific vibrational modes of deoxyribose moieties and presumably reflect desolvation at the nonpolar interface of protein and DNA. These Raman markers may be diagnostic of protein-induced DNA bending and are proposed as a baseline for comparative analysis of mutations in SRY that cause human sex reversal. The DNA-binding domains of gene-regulatory proteins exhibit considerable diversity in structural motif, mechanism of nucleotide recognition, and extent of induced fit at the protein-nucleic acid interface (1-3). Of particular interest is the phenomenon of protein-directed bending of the double helix, which may have functional consequences at sites re- mote from that of protein binding (4). Because high-reso- lution structures of DNA target sites in both protein-bound and protein-free states are not generally available, we are investigating the utility of Raman spectroscopy as a probe of protein-induced changes in DNA structure. The focus of the present study is on the high-mobility- group (HMG) 1 box of a putative transcriptional regulator encoded by the male sex-determining region of the human Y chrosomosome (hSRY). The 87-residue fragment inves- tigated here (5) is closely related to the hSRY fragment investigated previously by biochemical and NMR methods (6, 7). The hSRY-HMG box binds to the minor groove of its DNA target site and substantially distorts the B-DNA conformation, as depicted in Figure 1A. The mechanism in part involves the insertion of an isoleucyl side chain (Ile 13), which expands the minor groove and induces sharp bending of the double helix toward the compressed major groove (7, 8). The DNA-bound protein manifests the same L-shaped R-helical fold (Figure 1B) shared by the sequence-nonspecific class of HMG proteins (9-11). In contrast, the basic leucine zipper (bZIP) protein GCN4 recognizes the major groove but induces no appreciable change in DNA conformation upon binding to an AP-1 target site, as demonstrated in the following paper in this issue (12). Thus, DNA complexes of the hSRY-HMG box and GCN4 represent opposite extremes of a range of protein-directed DNA reorganizations studied by Raman spectroscopy (13-16). † Paper LXXII in the series Raman Spectral Studies of Nucleic Acids. Supported by NIH Grants GM54378 (G.J.T.), HD33462 (M.A.W.), and GM20861 (W.K.O.). M.A.W. was an American Heart Association Established Investigator and a Lucille Markey Scholar, University of Chicago. * To whom correspondence may be addressed. (G.J.T.) Phone: (816) 235-5247. E-mail: thomasgj@umkc.edu. (M.A.W.) Phone: (216) 368- 5991. E-mail: weiss@biochemistry.cwru.edu. ‡ University of MissourisKansas City. § University of Chicago. | Rutgers University. ⊥ Present address: Department of Biochemistry, Wood W427, Case Western Reserve School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4935. 1 Abbreviations: A260, absorbance at 260 nm; bp, base pair; bZIP, basic region of the leucine-zipper motif; CD, circular dichroism; FPLC, fast-protein liquid chromatography; HMG, high-mobility-group; HMQC, heteronuclear multiple quantum coherence; HSQC, heteronuclear single quantum coherence; hSRY, sex-determining region of the human Y chromosome; hSRY-HMG box, high-mobility-group box encoded by the male sex-determining region of the human Y chromosome; HTH, helix-turn-helix; MIS, Mu ¨llerian inhibiting substance; NMR, nuclear magnetic resonance; RMD, root-mean-distance; SDS, sodium dodecyl sulfate; TBP, TATA-box binding protein; UVRR, ultraviolet-resonance Raman spectroscopy. 537 Biochemistry 2000, 39, 537-547 10.1021/bi9900525 CCC: $19.00 © 2000 American Chemical Society Published on Web 12/22/1999