Elucidation of Lipid Binding Sites on Lung Surfactant Protein A Using X-ray Crystallography, Mutagenesis, and Molecular Dynamics Simulations

Abstract

Surfactant protein A (SP-A) is a collagenous C-type lectin (collectin) that is critical for pulmonary defense against inhaled microorganisms. Bifunctional avidity of SP-A for pathogen associated molecular patterns (PAMPs) such as lipid A and for dipalmitoylphosphatidylcholine (DPPC), the major component of surfactant membranes lining the air liquid interface of the lung, ensures that the protein is poised for first line interactions with inhaled pathogens. To better understand the motifs that are required for interactions with microbes and surfactant structures, we explored the role of the tyrosine-rich binding surface on the carbohydrate recognition domain of SP-A in the interaction with DPPC and lipid A using crystallography, site-directed mutagenesis, and molecular dynamics simulations. Critical binding features for DPPC binding include a three-walled tyrosine cage that binds the choline head group through cation-π interactions and a positively charged cluster that binds the phosphoryl group. This basic cluster is also critical for binding of lipid A, a bacterial PAMP and target for SP-A. Molecular dynamics simulations further predict that SP-A binds lipid A more tightly than DPPC. These results suggest that the differential binding properties of SP-A favor transfer of the protein from surfactant DPPC to pathogen membranes containing appropriate lipid PAMPs to effect key host defense functions.

Visualization of lipid directed dynamics of perilipin 1 in human primary adipocytes

“…Here, surfactant proteins (SP–A to –C) are abundantly expressed as membrane adherent proteins. Lipid binding studies of SP–A to phospholipids have shown that SP-A preferentially binds DPPC, the major lipid component of lung surfactant, but not to other phospholipids 60 , 61 . The native lung surfactant monolayer also exhibits topologically distinct spherical micro domains, as evidenced by atomic force microscopy 59 .…”

Interfacial Aromatics Mediating Cation−π Interactions with Choline-Containing Lipids Can Contribute as Much to Peripheral Protein Affinity for Membranes as Aromatics Inserted below the Phosphates

“…Both phosphatidylcholine (PC) and sphingomyelin, the major lipid components of eukaryotic plasma membranes, contain choline groups. We and others have observed cation-p interactions between peripheral proteins and membrane PC lipids (Cf Refs 17,[19][20][21][22][23][24][25] and Supporting Information). The energetics of cation-π interactions between various ions and aromatic groups have been estimated mostly in small model systems.…”

Lipid–Protein and Protein–Protein Interactions in the Pulmonary Surfactant System and Their Role in Lung Homeostasis

“…On the other hand, SP-C binds to the lipid A moiety of LPS by its N-terminal segment, probably through electrostatic interactions between the polar and basic residues of the protein and the negatively charged terminal 1-phosphate group at the reducing end of the lipid A disaccharide [199]. Similarly, SP-A binding to the 1-phosphate of lipid A [212] may involve electrostatic interactions with a cluster of basic residues located in the CRD of the protein [203,213]. Since the negative charges of the phosphate groups are required for LPS binding to LBP [214] and the activation of the TLR4/MD2 complex [215], the interaction of SP-C and SP-A with 1-phosphate may shield this functional group, blocking some of the physiological responses to LPS in the lungs.…”