Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

Area of Science:

• Plant molecular biology
• Protein structure-function relationships
• Cellular transport mechanisms

Background:

• The mechanisms by which sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release remain largely unknown.
• The plant vacuolar sorting receptor BP-80 is crucial for targeting polypeptide ligands containing a central Asn-Pro-Ile-Arg (NPIR) motif to the vacuole.
• Understanding BP-80's ligand recognition is vital for elucidating intracellular trafficking pathways in plants.

Purpose of the Study:

• To elucidate the structural basis of ligand recognition by the plant vacuolar sorting receptor BP-80.
• To determine how different domains of BP-80 contribute to the specific binding of polypeptide ligands.
• To investigate the role of the Asn-Pro-Ile-Arg (NPIR) motif and other determinants in BP-80-ligand interactions.

Main Methods:

• Purification and protease digestion of a soluble form of the BP-80 receptor (tBP-80).
• Ligand binding studies using full-length tBP-80 and a secreted protein lacking epidermal growth factor (EGF) repeats.
• Characterization of protease-resistant structural domains and their contribution to ligand binding specificity.

Main Results:

• tBP-80 binds polypeptide ligands with an NPIR motif as a monomer with high specificity.
• Three protease-resistant structural domains were identified: an N-terminal/ReMembR-H2 (RMR) homology domain, a central domain, and a C-terminal epidermal growth factor (EGF) repeat domain.
• The N-terminal/RMR and central domains together form the NPIR-specific binding site, while the C-terminal EGF repeat domain enhances ligand binding by altering conformation.
• A fragment containing the central and C-terminal domains binds ligand but lacks NPIR specificity, indicating two distinct binding sites.

Conclusions:

• The BP-80 receptor employs a multi-domain structure to achieve specific ligand recognition.
• Two distinct binding sites on tBP-80 recognize separate ligand determinants: an NPIR-specific site and a non-NPIR site.
• The interaction between the N-terminal/RMR and central domains is critical for NPIR motif recognition, while the overall domain structure influences ligand binding affinity and specificity.