Conversion of a human low-density lipoprotein receptor ligandbinding repeat to a virus receptor: identification of residues important for ligand specificity.
The amino-terminal region of the low-density lipoprotein receptor (LDLR) contains seven imperfect repeats of a cysteine-rich, roughly 40-aa module (LDL-A module) that are critical for apolipoprotein binding. LDL-A modules are found in numerous cell-surface and secreted proteins and are believed to mediate extracellular protein-protein interactions. The cellular receptor for subgroup A Rous sarcoma virus (RSV) contains a single LDL-A module that binds the RSV envelope protein and allows viral infection. To define residues in an LDL-A module responsible for ligand recognition, we used a gain of function assay by using a chimeric protein in which the LDL-A module of Tva was replaced with a highly homologous module from human LDLR (LDL-A4) and determined whether this chimera or mutants produced in it could mediate RSV infection. LDL-A4 does not function as an RSV receptor; however, systematic replacement of the nonconserved residues of the LDL-A4 module in the chimeric protein with the corresponding residues from Tva identified three residues sufficient to alter ligand specificity and convert LDL-A4 to an efficient viral receptor. Mutations of the corresponding residues in the Tva LDL-A module decreased both envelope binding and viral receptor function, confirming the importance of these residues in ligand recognition by this module. Analysis of the hLDL-A5 structure demonstrates that these three residues are clustered at one end of the LDL-A module. These results demonstrate that using a single LDL-A module in a gain of function assay is a useful model to investigate ligand recognition by this module.
Rong
L
L
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6076, USA.
Gendron
K
K
Bates
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011973:Receptors, LDL / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D008024:Ligands; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins