Arg343 in human surfactant protein D governs discrimination between glucose and N-acetylglucosamine ligands.
Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity.
Allen
Martin J
MJ
Department of Medicine, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA.
Laederach
Alain
A
Reilly
Peter J
PJ
Mason
Robert J
RJ
Voelker
Dennis R
DR
D000117:Acetylglucosamine / Q000737:chemistry*; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003198:Computer Simulation; D005947:Glucose / Q000737:chemistry*; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D037663:Pulmonary Surfactant-Associated Protein D / Q000737:chemistry* / Q000235:genetics
D008024:Ligands; D037663:Pulmonary Surfactant-Associated Protein D; D001120:Arginine; D005947:Glucose; D000117:Acetylglucosamine