Interactions between bovine seminal plasma proteins and model lipid membranes

1. Interactions between bovine seminal plasma proteins and model lipid membranes Danny Lassiseraye1, Puttaswamy Manjunath2 and Michel Lafleur1 1Département de chimie, Université de Montréal 2Centre de recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont

2. Introduction The bovine seminal plasma contains a family of proteins (BSP-A1/A2, BSP-A3 and BSP-30kDa, collectively called « BSP ») that bind to sperm membrane upon ejaculation. It has been shown that these proteins have a particular affinity for lipids bearing the choline moiety. The aim of our studies is to understand how these proteins interact with lipids. We examined the interactions between BSP proteins and lipid vesicles of different compositions. Isothermal titration calorimetry (ITC) has been used to determine the partition coefficients of the proteins with vesicles containing neutral lipids, charged lipids, and cholesterol. This technique also provided informations on the molar binding enthalpy, on the number of lipids interacting with one protein and on the effective charge of BSP upon binding. The effect of the BSP proteins on the morphology of the vesicles has been investigated with cryo-EM, showing a curvature effect on the vesicles and the formation of small vesicles. Finally, infrared spectroscopy indicated that the structure of the BSP proteins is stabilized by the presence of lipids.

3. Isothermal Titration Calorimetry ITC can measure « in situ » the enthalpy (DH) variation associated to a ligand binding to its substrate. BSP proteins are injected in the sample cell containing lipid vesicles. The binding enthalpy is detected by the variation of electrical power needed to maintain both cells at the same temperature.

4. Temperature effect on the binding of BSP proteins to POPC vesicles 10°C 20°C 37°C

5. Temperature effect on the binding of BSP proteins to POPC/POPG vesicles 37°C 20°C

6. Temperature effect on the binding of BSP proteins to POPC/Chol vesicles 20°C 37°C

7. ITC Results Table 1 : Membrane saturation (lipids/BSP) and molar binding enthalpy for the titration of four lipid systems with BSP proteins. Saturation ratios are calculated taking into account only the outer leaflet of the lipid vesicles. Table 2 : Partition coefficients and effective charge for the binding of BSP proteins to the four studied systems. a : Value only attributed to the transfer of the protein in the lipid membrane. b : Value including the electrostatic interactions and the transfer of the protein in the lipid membrane

8. Conclusions - ITC • Affinity : PC/PE > PC/PG > PC > PC/Chol. • Temperature affects the titration profiles. • A multimer-dimer transition was reported for the BSP proteins at 36°C. • However, considerable differences were observed as a function of membrane composition. • Thus, the resulting behavior appears to reflect both the solution structure of BSP proteins and variation of membrane composition. • The saturation molar binding ratio (Lipid/BSP) changes with the lipid composition of vesicles. • The presence of a negative charge on the membrane leads to higher molar binding enthalpy (DH), suggesting electrostatic interactions.

9. 60 °C 20 °C 1640 1600 1700 1680 1620 1660 Wavenumber (cm-1) Infrared Spectroscopy Spectra series of free BSP in solution, showing the evolution of the Amide I band between 20 and 60°C. The maximum of the band is shifted towards higher frequencies.

10. Amide I band shift of free and lipid-bound BSP The presence of lipids affects the temperature at wich the maximum of the Amide I band is shifted. The binding to lipids seems to stabilize the protein structure.

11. Cryo-EM POPC unilamellar vesicles Addition of BSP (Ri 48) to POPC LUV

12. Cryo-EM Addition of BSP (Ri 48) to PC/PE LUV Addition of BSP (Ri 48) to PC/Chol LUV

13. Conclusions – Cryo-EM • Addition of BSP modifies the morphology of the vesicles. • Formation of tube-like structures, of stretched and connected structures, and budding. • Tube-like structures seem to have a smaller diameter with the PC/PE system. • Stretched and connected structures seem to be more abundant with the PC/Chol system.

14. General Conclusions • The lipid/BSP saturation ratio correlates with the order of the bilayer: PC/Chol > PC/PE > PC ≈ PC/PG. • This may be related to the morphology changes of the vesicles upon BSP proteins binding. • The binding to lipid membranes stabilizes the BSP proteins solution structure. • Temperature affects the binding of BSP proteins to vesicles of different lipid compositions.