Equilibrium Unfolding of the C-Terminal SAM Domain of p73

Abstract

The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the folding equilibrium properties of the p73 alpha SAM domain (SAMp73) by using different biophysical techniques (circular dichroism, fluorescence, and Fourier transform infrared spectroscopies, and differential scanning calorimetry). Those probes indicate that SAMp73 folds via a two-state mechanism. Fluorescence experiments performed at different pHs showed two titrations: the first one due to an acid residue (with a pK(a) = 4.5 +/- 0.3) and the second due to deprotonation of tyrosine residues. The conformational stability of the protein upon chemical denaturation was determined over the pH range 3 to 10. The maximum conformational stability is DeltaG = 5.7 +/- 0.4 kcal x mol(-1) (at 25 degrees C) and occurs in a broad maximum, with little variation, between pH 6 and 10. The high melting temperature of SAMp73 (T(m) = 93.5 degrees C), despite its moderate conformational stability at 25 degrees C, can be ascribed to its low heat capacity change upon unfolding, DeltaC(p), which is estimated to be around 915 cal x K(-1) x mol(-1) at 25 degrees C and only around 543 cal x K(-1) x mol(-1) at the T(m). The implications of the temperature-dependent nature of DeltaC(p) are discussed in relation to the thermal stability of proteins as opposed to their conformational stability at room temperature.

Binding of the C-terminal Sterile α Motif (SAM) Domain of Human p73 to Lipid Membranes

“…Trp 542 , in the numbering of intact p73␣, is in the middle of helix 5 forming the hydrophobic core of the protein; Tyr 487 is the N-terminal residue; Tyr 508 is in the middle of helix 2; Tyr 518 and Tyr 537 are at the beginnings of helices 3 and 5, respectively; and Tyr 554 is the C-terminal residue. The emission fluorescence spectrum of native SAMp73 is dominated by the emission of the sole tryptophan residue (18), with a maximum at 336 nm at neutral pH, which indicates that the tryptophan is partially buried within the protein structure, as concluded from the x-ray (15,16) and NMR structures (14).…”

“…Recently, however, the SAM domain of p73, SAMp73, has been shown to be monomeric in solution (14,18). Furthermore, although the SAM domains of EphA4 and B2 receptors formed oligomers, oligomerization was not observed for both receptors (17,46).…”

“…Because SAMp73 is a highly compact protein, which buries a large amount of hydrophobic surface (14,18), how can SAMp73 bind lipids? Which are the protein-interacting regions?…”

“…Differences in the heat capacity between the sample and the reference cell, filled with buffer solution, were obtained by raising the temperature at a constant rate of 60°C/h over a temperature range of 10 -60°C. At these temperatures thermal unfolding of the protein is not expected (18). The excess heat capacity functions were obtained after base-line subtraction and correction for the instrument time response.…”

“…SAM domains are putatively considered to be responsible for regulating protein functions via self-association or by association with other domains (17), but the exact function of SAMp73 is not known. The crystal structure of SAMp73 reveals a dimeric organization (15), but the NMR structure is monomeric (14), suggesting that dimer formation in the crystal is an effect of crystal packing rather than a real physiological state; furthermore, equilibrium sedimentation experiments have shown that SAMp73 is monomeric under a wide range of experimental conditions (14,18).…”