Revised Manuscript Received July 18, 1995s abstract: The conformation of an eight base pair DNA oligonucleotide duplex bound tothe human testis determiningfactor SRY and the orientation of the protein domain within the complex have been analyzed by a variety of NMR methods which permit the selective observation of protons attached to 12C nuclei in thepresence of uniformly enriched 13C/15N protein. Qualitative analysis of nuclear and rotating frame Overhauser enhancement spectra at multiple mixing times indicates that the conformation of the SRY-bound DNA is distinct from that of A-and B-DNA, in agreement with the recent three-dimensional structure determination of the complex [Wemer, M. W., Huth, J. R., Gronenbom, AM, & Clore, G. M.(1995) Cell 81, 705—714], Selective observation of intermolecular NOEs between protein and DNA indicates that partial intercalation of a protein side chain occurs between two adenine bases in the DNA octamer. The analysis of structural features by NMR for this unusual DNA conformer and the orientation of the protein domain on the DNA is discussed. The structural features of the DNA complexed to SRY are remarkably similar, but not identical, to those of DNA complexed to the TATA-binding protein (TBP).

Human SRY (hSRY) protein, also known as the human testis determining factor, is a Y-chromosome encoded transcription factor that acts as a genetic switch in gonadal differentiation (Goodfellow & Lovell-Badge, 1993; Gustafson et al., 1994; McElreavey et al., 1993). The DNA-binding domain of hSRY is a member of the HMG-1/2 superfamily of DNA-binding proteins (Sinclair et al., 1990), and the structure of this domain complexed to an eight base pair oligonucleotide duplex derived from the human Mullerian inhibitor substance (MIS) 1 promoter has recently been solved by multidimensional heteronuclear NMR spectroscopy (Wem-er et al., 1995). Structural and biochemical data suggest that the primary function of hSRY is its ability to dramatically distort the DNA conformation, in particular to cause helix unwinding, minor groove expansion, and bending (Wemer et al., 1995). With the exception of SRY-bound DNA, all DNA duplexes analyzed by NMR, either free in solution or in complexes with proteins, have been of the B type. While the pattern of nuclear Overhauser effects for B-and A-DNA are well established [see Clore and Gronenbom (1985) and van De Ven and Hilbers (1988) for reviews], those for a highly distorted conformation such as that found in the SRY-bound DNA are not. In this paper, we therefore present a qualitative analysis of the spectroscopic characteristics of