Investigating the structural basis of surfactant protein D oligomerization: The crucial role of Cys15-Cys15/Cys20-Cys20 disulfide bond linkage.

Surfactant protein D (SP-D) plays a vital role in innate immunity by recognizing and binding to glycans on the surfaces of various pathogens. The oligomerization of SP-D into dodecameric structures enhances its pathogen-binding and antiviral activities. Central to this oligomerization are two conserved cysteine residues (Cys15 and Cys20) in the N-terminal domain, which form disulfide bonds essential for assembly. However, the precise disulfide bond linkages within the N-terminal domain remain unclear, limiting our understanding of SP-D's assembly mechanism at the molecular level. In this study, we investigated the four possible disulfide linkage types of the SP-D N-terminal domain to elucidate their effects on structure. Using replica exchange molecular dynamics (REMD) simulations, we extensively sampled the conformational space of each linkage type to identify predominant conformations. These representative structures were used to model SP-D dodecamer assembly. Extensive unbiased molecular dynamics (MD) simulations with three independent replicates per linkage type assessed the structural stability of each dodecamer model. Our results indicate that the disulfide linkage, where disulfide bonds form between Cys15/Cys20 residues of adjacent monomers, leads to a stable dodecamer structure most consistent with experimental observations from atomic force microscopy (AFM). In contrast, the other three linkage types showed greater structural instability and did not align with experimental data. These findings provide molecular-level insights into SP-D assembly, suggesting that the disulfide linkage between Cys15/Cys20 residues is strongly supported as the physiologically relevant configuration for dodecamer formation, enhancing our understanding of SP-D's role in innate immunity and informing future therapeutic strategies targeting SP-D function.