Backbone dynamics of sequence specific recognition and binding by the yeast Pho4 bHLH domain probed by NMR

Backbone dynamics of the basic/helix-loop-helix domain of Pho4 from Sacchar omyces cerevisiae have been probed by NMR techniques, in the absence of DNA . nonspecifically bound to DNA and bound to cognate DNA. Alpha proton chemi cal shift indices and nuclear Overhauser effect patterns were used to eluci date the secondary structure in these states. These secondary structures ar e compared to the co-crystal complex of Pho4 bound to a cognate DNA sequenc e (Shimizu T Toumoto A, Ihara K, Shimizu M, Kyogou Y, Ogawa N, Oshima Y, Ha koshima T, 1997, EMBO J 15: 4689-4697). The dynamic information provides in sight into the nature of this DNA binding domain as it progresses from free in solution to a specifically bound DNA complex. Relative to the unbound f orm, we show that formation of either the nonspecific and cognate DNA bound complexes involves a large change in conformation and backbone dynamics of the basic region. The nonspecific and cognate complexes, however, have nea rly identical secondary structure and backbone dynamics. We also present ev idence for conformational flexibility at a highly conserved glutamate basic region residue. These results, art: discussed in relation to the mechanism of sequence specific recognition and binding.