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.
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(S(i)), 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.
Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche (Alicante), Spain. jlneira@umh.es
José L
JL
Neira
Paz
P
Sevilla
Francisco
F
García-Blanco
D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D008958:Models, Molecular; D009068:Movement / Q000187:drug effects; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D055550:Protein Stability / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013696:Temperature; D013997:Time Factors; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; D019791:Guanidine