Acidic Environment Induces Dimerization and Ligand Binding Site Collapse in the Vps10p Domain of Sortilin

Abstract

Sortilin is a neuronal receptor involved in transmembrane signaling, endocytosis, and intracellular sorting of proteins. It cycles through a number of cellular compartments where it encounters various acidic conditions. The crystal structure of the sortilin ectodomain has previously been determined at neutral pH. Here, we present the 3.5-Å resolution crystal structure of sortilin at pH 5.5, which represents an environment similar to that of late endosomes, where ligands are released. The structure reveals an overall distortion of the 10-bladed β-propeller domain. This distortion and specific conformational changes, caused by protonation of a number of histidine residues, render the currently known binding sites unavailable for ligand binding. Access to the binding sites is furthermore blocked by a reversible and pH-dependent formation of tight sortilin dimers, also confirmed by electron microscopy, size-exclusion chromatography, and mutational studies. This study reveals how sortilin binding sites are disrupted and explains pH-dependent ligand affinity.

Crystal structure of the ligand‐free form of the Vps10 ectodomain of dimerized Sortilin at acidic pH

“…Therefore, the pH at the cellular compartments containing Sortilin dramatically changes during Sortilin trafficking in cells. Consistent with the findings of the most recent studies [25,26], we also demonstrated that sSortilin dimerizes under a low pH buffer condition. In addition, SEC analysis revealed that the A495E mutant in mouse Sortilin, which is located in the vicinity of F128 at the hydrophobic loop, abolishes the potency of pH-dependent dimerization [26].…”

“…Consequently, our structure could clearly represent the contribution of E455 to the hydrophobic interaction with F137 in Sortilin dimerization at acidic pH. The conformational changes that were observed upon dimerization, as revealed through structural comparison with human sSortilin, were also confirmed independently in these recent studies [25,26], indicating the importance of conformational changes for dimerization of sSortilin at acidic pH.…”

“…During preparation of this paper, the crystal structures of the human and mouse sSortilin dimer were reported by two independent groups [25,26]; however, the crystallization conditions employed in these studies were quite different from those in the present study. Consequently, the cell dimensions of their crystals also differed from those of our crystals.…”

“…Consequently, the cell dimensions of their crystals also differed from those of our crystals. Structural alignment revealed that our determined sSortilin dimer well resembled both mouse sSortilin dimer (PDB ID: 5NMT) [26] (the Ca-Ca RSMD is 0.93 A) and human sSortilin dimer (PDB ID: 6EHO) [25] (the Ca-Ca RSMD is 0.91 A) ( Fig. S1A, B).…”

“… indicated that the N272Q mutant could not be produced due to its very low expression levels, which suggested that the N‐linked glycan on N272 functions in maintaining the structural stability and/or appropriate intracellular trafficking of sSortilin in addition to its role in dimerization. Given that SEC analysis demonstrated that both the glycosylated and deglycosylated samples of human sSortilin could dimerize in acidic pH buffer , the glycosylation does not appear to be critical for the dimerization, although further experiments are required to elucidate the precise role of glycans in pH‐dependent Sortilin dimerization. Collectively, our findings highlight the importance of the pH‐dependent dimerization and N‐glycosylation of Sortilin in cytokine trafficking in cells.…”