Lung surfactant protein A (SP-A) interactions with model lung surfactant lipids and an SP-B fragment

Abstract

Surfactant protein A (SP-A) is the most abundant protein component of lung surfactant, a complex mixture of proteins and lipids. SP-A performs host defense activities and modulates the biophysical properties of surfactant in concerted action with surfactant protein B (SP-B). Current models of lung surfactant mechanism generally assume SP-A functions in its octadecameric form. However, one of the findings of this study is that when SP-A is bound to detergent and lipid micelles that mimic lung surfactant phospholipids, it exists predominantly as smaller oligomers, in sharp contrast to the much larger forms observed when alone in water. These investigations were carried out in sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), lysomyristoylphosphatidylcholine (LMPC), lysomyristoylphosphatidylglycerol (LMPG), and mixed LMPC + LMPG micelles, using solution and diffusion nuclear magnetic resonance (NMR) spectroscopy. We have also probed SP-A's interaction with Mini-B, a biologically active synthetic fragment of SP-B, in the presence of micelles. Despite variations in Mini-B's own interactions with micelles of different compositions, SP-A is found to interact with Mini-B in all micelle systems and perhaps to undergo a further structural rearrangement upon interacting with Mini-B. The degree of SP-A-Mini-B interaction appears to be dependent on the type of lipid headgroup and is likely mediated through the micelles, rather than direct binding.

Figure 1

HN regions of 1D ¹H NMR spectra of SP-A in water and in different micelle environments. (A) 0.2 mM SP-A in water and in 40 mM SDS and 40 mM DPC (256 scans). (B) 0.25 mM SP-A in 50 mM LMPC, 50 mM LMPG, and 42.5 mM LMPC + 7.5 mM LMPG (160 scans). All spectra within each panel are shown with the same intensity scale. However, the intensity scales are not comparable between the panels as sample compositions and acquisition parameters were different.

Figure 4

2D ¹⁵N–¹H HSQC spectra of Mini-B in different micelles in the absence (top panels) and presence (bottom panels) of SP-A. 0.2 mM Mini-B (A) and 0.1 mM Mini-B + 0.1 mM SP-A (F) in 40 mM SDS. 0.2 mM Mini-B (B) and 0.1 mM Mini-B + 0.1 mM SP-A (G) in 40 mM DPC. 0.25 mM Mini-B (C) and 0.125 mM Mini-B + 0.125 mM SP-A (H) in 50 mM LMPC. 0.25 mM Mini-B (D) and 0.125 mM Mini-B + 0.125 mM SP-A (I) in 50 mM LMPG. 0.25 mM Mini-B (E) and 0.125 mM Mini-B + 0.125 mM SP-A (J) in 42.5 mM LMPC + 7.5 mM LMPG. Spectra A–E were acquired using 160 scans, and spectra F–J were acquired using 320 scans.

Figure 2

Translational diffusion measurements of SP-A in water, SDS, and DPC micelles. Top panels show the 2D DOSY spectra of 0.2 mM SP-A in water (A), 0.2 mM SP-A in 40 mM SDS (B), and 0.2 mM SP-A in 40 mM DPC (C). Bottom panels show the linear fits obtained for the attenuation of the integrated HN region of SP-A in water (D), in complex with SDS (E), and in complex with DPC (F). The linear fits for pure SDS (40 mM) and DPC (40 mM) micelles, obtained from the attenuation of the peak at 0.80 ppm, are included in (E) and (F) for comparison.

Figure 3

Translational diffusion measurements of SP-A and Mini-B in LMPC, LMPG, and LMPC (85%) + LMPG (15%) micelle systems. 2D DOSY data were acquired separately for pure micelles (50 mM), SP-A (0.25 mM) in micelles (50 mM), Mini-B (0.25 mM) in micelles (50 mM), and SP-A (0.125 mM) + Mini-B (0.125 mM) in micelles (50 mM). Linear fits show the attenuation of the ¹H signals for micelles and protein–micelle complexes as determined from the lipid peak at 0.86 ppm.

Figure 5

Signal attenuation curves obtained from the translational diffusion measurements of 0.1 mM SP-A + 0.1 mM Mini-B in 40 mM SDS (A) and in 40 mM DPC (B). None of the curves fit well with a single line. However, approximately the first and the last halves of the data fit well with two lines having two different slopes. Consequently, two diffusion coefficients are obtained for each system.

Figure 6

Comparison of the average apparent hydrodynamic diameters (d_HA) of SP-A in water, pure micelles, individual SP-A– and Mini-B–micelle complexes, and combined SP-A–Mini-B–micelle complexes as calculated from the 2D DOSY NMR spectra.