Analysis of the Structural Organization and Thermal Stability of two Spermadhesins

The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel β-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgroup of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organzation of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-TI) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5°C and 78.6°C, respectively. The calorimetric enthalpy changes (ΔHcal) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that β-structure and turns are the major structural element of both PSP-I/PSP-II (53% of β-sheet, 21% of turns) and aSFP (44% of β-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, α-helical structure. Taken together, Our results indicate that the porcine and the bovine spermadhesin molecules are probably all-β-structure proteins, and would support a β-barrel topology like that predicted for the CUB domain. Other β-structure folds, such as the Greek-key pattern characteristic of many carbohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms.