Contributions of Phenylalanine 335 to Ligand Recognition by Human Surfactant Protein D: RING INTERACTIONS WITH SP-D LIGANDS

Erika Crouch,Barbara Mcdonald,Kelly Smith,Tanya Cafarella,Barbara Seaton,James Head

doi:10.1074/jbc.m601749200

Abstract

Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Interactions of SP-D with microorganisms and organic antigens involve binding of glycoconjugates to the C-type lectin carbohydrate recognition domain (CRD). A trimeric fusion protein encoding the human neck+CRD bound to the aromatic glycoside p-nitrophenyl-alpha-D-maltoside with nearly a log-fold higher affinity than maltose, the prototypical competitor. Maltotriose, which has the same linkage pattern as the maltoside, bound with intermediate affinity. Site-directed substitution of leucine for phenylalanine 335 (Phe-335) decreased affinities for the maltoside and maltotriose without significantly altering the affinity for maltose or glucose, and substitution of tyrosine or tryptophan for leucine restored preferential binding to maltotriose and the maltoside. A mutant with alanine at this position failed to bind to mannan or maltose-substituted solid supports. Crystallographic analysis of the human neck+CRD complexed with maltotriose or p-nitrophenyl-maltoside showed stacking of the terminal glucose or nitrophenyl ring with the aromatic ring of Phe-335. Our studies indicate that Phe-335, which is evolutionarily conserved in all known SP-Ds, plays important, if not critical, roles in SP-D function.

Highlights

Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation
Because maltose is the most potent simple competitor of SP-D binding to various sugar ligands, we examined the binding of human SP-D NCRDs to p-nitrophenyl-␣-D-maltoside
The mutagenesis and crystallographic data indicate that the aromatic ring of phenylalanine 335 (Phe-335) provides a secondary stabilizing interaction that renders maltotriose more effective as a competitor than the prototypical saccharide ligand, maltose

Summary

Introduction

Surfactant protein D (SP-D) is an innate immune effector that contributes to antimicrobial host defense and immune regulation. Surfactant Protein D (SP-D) is a collagenous C-type lectin (collectin) that contributes to antimicrobial host defense and immune regulation in the lung and certain extrapulmonary tissues [1,2,3,4]. Crystallographic studies of trimeric human SP-D neckϩCRD domains have shown that maltose, a preferred saccharide ligand, binds to calcium via the vicinal 3- and 4-OH groups of the non-reducing glucose, previously designated calcium ion 1 and glucose 1 (Glc1), respectively [8]. These interactions are further stabilized by hydrogen bonding of Glc to amino acid side chains that coordinate with calcium ion 1

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Conclusion