Folding of a dimeric beta-barrel: Residual structure in the urea denaturedstate of the human papillomavirus E2 DNA binding domain

The dimeric beta-barrel is a characteristic topology initially found in the transcriptional regulatory domain of the E2 DNA binding domain from papill omaviruses. We have previously described the kinetic folding mechanism of t he human HPV-16 domain, and, as part of these studies, we present a structu ral characterization of the urea-denatured state of the protein. We have ob tained a set of chemical shift assignments for the C-terminal domain in ure a using heteronuclear NMR methods and found regions with persistent residua l structure. Based on chemical shift deviations from random coil values, (3 )J(NHN alpha) coupling constants, heteronuclear single quantum coherence pe ak intensities, and nuclear Overhauser effect data, we have determined clus ters of residual structure in regions corresponding to the DNA binding heli x and the second beta-strand in the folded conformation. Most of the struct ures found are of nonnative nature, including turn-like conformations. Urea denaturation at equilibrium displayed a loss in protein concentration depe ndence, in absolute parallel to a similar deviation observed in the folding rate constant from kinetic experiments. These results strongly suggest an alternative folding pathway in which a dimeric intermediate is formed and t he rate-limiting step becomes first order at high protein concentrations. T he structural elements found in the denatured state would collide to yield productive interactions, establishing an intermolecular folding nucleus at high protein concentrations. We discuss our results in terms of the folding mechanism of this particular topology in an attempt to contribute to a bet ter understanding of the folding of dimers in general and intertwined dimer ic proteins such as transcription factors in particular.