The C-terminal sterile alpha motif (SAM) domain of human p73 is a highly dynamic protein, which acquires high thermal stability through a decrease in backbone flexibility

Abstract

The α-splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has close to its C terminus a sterile alpha motif (SAM), SAMp73, that is involved in protein–biomolecule interactions. The conformational stability of SAMp73 is low (∼5 kcal mol⁻¹), although its thermal stability is high. To explain this high thermostability, we studied the dynamics of SAMp73 over a wide range of GdmCl (guanidine hydrochloride) concentrations and temperatures by NMR relaxation, NMR hydrogen-exchange (HX) and fluorescence lifetime approaches. The slowest exchanging residues of SAMp73 belong to the helical regions, and they did exchange by a global unfolding process. Moreover, SAMp73 was very flexible, with most of its amide protons affected by slow μs–ms conformational exchange. Within this time scale, the residues of SAMp73 with the largest exchange rates (R_ex) were involved in binding with other molecules; therefore, the flexibility in the μs–ms range was associated with biological functions. As the [GdmCl] increased, the pico-to-nanosecond flexibility of the backbone amide protons raised, but it did so differently depending on the residue. We were able to obtain, for the first time, the linear [GdmCl]-variation of the local conformational entropies, m_Si, which ranged from 5.3 to 0.3 cal mol⁻¹ K⁻¹ M⁻¹, similar to those measured by using macroscopic techniques in other proteins. Conversely, the temperature dependence of the pico-to-nanosecond dynamics of the backbone amide protons of SAMp73 indicates that the flexibility of some residues decreased with the temperature; these results explain the high thermostability of the protein.