Differential ligand binding specificities of the pulmonary collectins are determined by the conformational freedom of a surface loop.

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 dipalmitoylphosphatidyleholine :(DPPC), the major surfactant lipid component, but not phosphatidylinositol (PI), whereas SP-D shows the opposite preference. Additionally, SP-A and SR-Drecognize 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 hypcithesis 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) thatintroduced an SP-D-like loop-stabilizing, calcium binding site into the carbohydrate recognition domain was found to interconvertsR,A ligand binding preferences to an SP-D' phenotype, exchanging DPPC for PI specificity, and tesulting 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 propertiesthat 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.