Structural requirements for ligand binding by a probable plant vacuolar sorting receptor.
How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.
Cao
X
X
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
Rogers
S W
SW
Butler
J
J
Beevers
L
L
Rogers
J C
JC
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004331:Drosophila melanogaster; D010450:Endopeptidases / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018532:Pisum sativum / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020449:Repetitive Sequences, Amino Acid; D017384:Sequence Deletion / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D033921:Vesicular Transport Proteins
D000911:Antibodies, Monoclonal; D004220:Disulfides; D004792:Enzyme Precursors; D005456:Fluorescent Dyes; D008024:Ligands; D010446:Peptide Fragments; D010940:Plant Proteins; D011956:Receptors, Cell Surface; D033921:Vesicular Transport Proteins; D004815:Epidermal Growth Factor; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; C076060:proaleurain