Structural basis for SRY-dependent 46-X,Y sex reversal: Modulation of DNA bending by a naturally occurring point mutation

The HMG-box domain of the human male sex-determining factor SIZY, hSRY(HMG) (comprising residues 57-140 of the full-length sequence), binds DNA sequen ce-specifically in the minor groove, resulting in substantial DNA bending. The majority of point mutations resulting in 46X,Y sex reversal are located within this domain. One clinical de novo mutation, M64I in the full-length hSRY sequence, which corresponds to M9I in the present hSRYHMG construct, acts principally by reducing the extent of DNA bending. To elucidate the st ructural consequences of the M9I mutation, we have solved the 3D solution s tructures of wild-type and M9I hSRY(HMG) complexed to a DNA 14mer by NMR, i ncluding the use of residual dipolar couplings to derive long-range orienta tional information. We show that the average bend angle (derived from an en semble of 400 simulated annealing structures for each complex) is reduced b y similar to 13 degrees from 54(+/-2)degrees in the wild-type complex to 41 (+/-2)degrees in the M9I complex. The difference in DNA bending can be loca lized directly to changes in roll and tilt angles in the ApA base-pair step involved in interactions with residue 9 and partial intercalation of Ile13 . The larger bend angle in the wild-type complex arises as a direct consequ ence of steric repulsion of the sugar of the second adenine by the bulky S- delta atom of Met9, whose position is fixed by a hydrogen bond with the gua nidino group of Arg17. In the M9I mutant, this hydrogen bond can no longer occur, and the less bulky C(gamma)m methyl group of Ile9 braces the sugar m oieties of the two adenine residues, thereby decreasing the roll and tilt a ngles at the ApA step by similar to8 degrees and similar to5 degrees, respe ctively, and resulting in an overall difference in bend angle of similar to 13 degrees between the two complexes. To our knowledge, this is one of the first examples where the effects of a clinical mutation involving a protei n-DNA complex have been visualized at the atomic level.