Inter-Monomer Interactions are Essential for Lysosomal Enzyme Binding by the Cation-dependent Mannose 6-Phosphate Receptor

• Published in: Biochemistry (Easton) Vol. 49; no. 1; pp. 236 - 246
• Main Authors: Olson, Linda J., Sun, Guangjie, Bohnsack, Richard N., Peterson, Francis C., Dahms, Nancy M., Kim, Jung-Ja P.
• Format: Journal Article
• Language: English
• Published: 12.01.2010
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi901725x

The 46 kDa cation-dependent MPR (CD-MPR) plays a key role in the delivery of lysosomal enzymes to the lysosome by binding newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and diverting them from the secretory pathway. Previous studies on a truncated form of the receptor comprised of only the soluble extracellular region (sCD-MPR, residues 1-154) have shown that the CD-MPR exists as a homodimer and exhibits two distinct conformations in the ligand-bound versus ligand-unbound states, involving changes in quaternary structure and positioning of loop D, the residues of which form a side of the binding pocket in the presence of ligand. To determine the role of inter-monomer contacts in the functioning of the sCD-MPR, site-directed mutagenesis was used to generate a construct lacking a salt bridge (Glu19-Lys137) that tethers the N-terminal α-helix of one subunit to loop D of the other subunit in the ligand-bound form. Here we show by surface plasmon resonance analyses and NMR spectroscopy that the elimination of this inter-monomer salt bridge significantly decreases binding affinity of the mutant receptor (E19Q/K137M) towards lysosomal enzymes and Man-6-P. Analyses of the E19Q/K137M mutant receptor crystallized under various conditions revealed an altered quaternary structure that is intermediate between those observed in the ligand-bound and ligand-unbound states. Taken together, the results demonstrate a key role for inter-monomer interactions in the structure and functioning of the CD-MPR.