Differential Ligand Binding Specificities of the Pulmonary Collectins Are Determined by the Conformational Freedom of a Surface Loop

Abstract

Lung surfactant proteins (SPs) play critical roles in surfactant function and innate immunity. SP-A and SP-D, members of the collectin family of C-type lectins, exhibit distinct ligand specificities, effects on surfactant structure, and host defense functions despite extensive structural homology. SP-A binds to dipalmitoylphosphatidylcholine (DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SP-D recognize widely divergent pathogen-associated molecular patterns. Previous studies suggested that a ligand-induced surface loop conformational change unique to SP-A contributes to lipid binding affinity. To test this hypothesis and define the structural features of SP-A and SP-D that determine their ligand binding specificities, a structure-guided approach was used to introduce key features of SP-D into SP-A. A quadruple mutant (E171D/P175E/R197N/K203D) that introduced an SP-D-like loop-stabilizing calcium binding site into the carbohydrate recognition domain was found to interconvert SP-A ligand binding preferences to an SP-D phenotype, exchanging DPPC for PI specificity, and resulting in the loss of lipid A binding and the acquisition of more avid mannan binding properties. Mutants with constituent single or triple mutations showed alterations in their lipid and sugar binding properties that were intermediate between those of SP-A and SP-D. Structures of mutant complexes with inositol or methyl-mannose revealed an attenuation of the ligand-induced conformational change relative to wild-type SP-A. These studies suggest that flexibility in a key surface loop supports the distinctive lipid binding functions of SP-A, thus contributing to its multiple functions in surfactant structure and regulation, and host defense.

The Role of Collectins and Galectins in Lung Innate Immune Defense

“…Their globular domains recognize not only carbohydrates but also a broader repertoire of ligands, including proteins, nucleic acids, and lipids ( 7 , 18 , 19 ). Despite their similar CRDs, SP-A and SP-D show significant differences in ligand preferences, with SP-A ligands generally being more amphipathic ( 19 , 20 ), and SP-D ligands richer in carbohydrates ( 20 ). These different preferences likely serve to extend the range of innate immune surveillance in the lung.…”

Role of inositol to improve surfactant functions and reduce IL-6 levels: A potential adjuvant strategy for SARS-CoV-2 pneumonia?

“…The protein component of surfactant includes the hydrophobic surfactant proteins (SPs)-B and -C, and the hydrophilic SPs-A and -D [4] . In the alveoli, these proteins are organized with the lipid components in mono- and multilayered aggregate membranes as micelles [5] .…”

Structural and Functional Determinants of Rodent and Human Surfactant Protein A: A Synthesis of Binding and Computational Data

“…Binding to extracellular ligands depends on the neck-CRD trimer and high avidity multivalent binding of the SP-A deca-octamer. Crystallographic and molecular modeling studies of the rat SP-A CRD revealed that conformational flexibility of the Ca ++ coordination site and CRD surface loops impart versatility in the ability of SP-A to bind structurally diverse endogenous and pathogen-derived ligands (2, 4–7). The interaction of SP-A with AMs and epithelial cells is receptor-mediated (8–10).…”

“…Deletion, site-directed mutagenesis, and ligand competition studies showed that the amino-terminal and collagen-like domains influence receptor occupancy and functional responses in alveolar type II epithelial cells and macrophages, and interaction of the CRD domain with surfactant phospholipids (10–16). SP-A binds dipalmitoyl phosphatidylcholine, the major surfactant phospholipid, that is required for the formation of tubular myelin from secreted lamellar bodies, contributes to the adsorption of surface active phospholipids at the air-liquid interface, and facilitates turnover of spent surfactant vesicles by alveolar type II epithelial cells through the p63 receptor (7, 17–21). In host defense, SP-A binds different pathogen ligands such as the lipid A portion of lipopolysaccharide on Gram-negative bacteria (7), surface proteins and glycolipids on gram positive bacteria (22), mycobacteria (23, 24), and fungi (25, 26).…”

“…SP-A binds dipalmitoyl phosphatidylcholine, the major surfactant phospholipid, that is required for the formation of tubular myelin from secreted lamellar bodies, contributes to the adsorption of surface active phospholipids at the air-liquid interface, and facilitates turnover of spent surfactant vesicles by alveolar type II epithelial cells through the p63 receptor (7, 17–21). In host defense, SP-A binds different pathogen ligands such as the lipid A portion of lipopolysaccharide on Gram-negative bacteria (7), surface proteins and glycolipids on gram positive bacteria (22), mycobacteria (23, 24), and fungi (25, 26). These interactions facilitate pathogen clearance through agglutination, opsonization, and direct killing (22, 26–29).…”