Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx

Abstract

Background: The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds.

Results: The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy.

Conclusion: The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.

Figure 1

The LAMP protein family. (A) Domain architectures of the five human LAMP family proteins (UniProt P11279, P13473, Q9UQV4, Q9UJQ1, P34810). O- and N-linked glycosylation sites are indicated in blue and red, respectively. SP, signal peptide; TM, transmembrane helix. (B, C) Sequence alignments of the seven human LAMP domains and adjacent transmembrane domains (B) and of the 'hinge' region (linker) connecting the membrane-distal ('dist') and membrane-proximal ('prox') domains of LAMP-1 and LAMP-2 (C). Glycosylated residues are displayed with yellow background. Residues involved in β-bulges are indicated by arcs in magenta. Hydrophobic residues are colored red and hydrophilic ones are colored blue (in B). S-S: disulfide bond.

Figure 2

The structure of the DC-LAMP domain. (A) Different views of the domain's shape. The two β-sheets are drawn in red and blue. (B) Stereo views of the domain's β-prism shape. Schematic prism shapes are drawn on the right for orientation. The N-acetyl-glucosamine residue is depicted as a stick drawing in cyan.

Figure 3

The β-strand arrangement of the DC-LAMP domain. The 'front' and 'back' β-sheets are drawn in red and blue, respectively. The cysteines that form the first disulfide bond (S-S) are labelled. β-strands and loops are identified as S1 to S11 and L1 to L3, respectively. The topology of the β-sheets is drawn schematically in the lower part with sheets opened out.

Figure 4

Stereo views of the β-bulges. The β-bulges that bend the front and back β-sheets are shown. Main chain atoms of parts of β-strands are shown as stick drawings. Bulged residues are numbered and their conformations are given in parentheses (α, α-helix; β, β-strand; I', type I' β-turn; Lα, left-handed α-helix). Hydrogen bond pairs that flank β-bulges are shown in red and the corresponding residues are labelled. Other inter-main chain hydrogen bonds are drawn in grey.

Figure 5

The hydrophobic pocket and the variable loop conformation. (A) The hydrophobic pocket in the DC-LAMP domain was filled with water molecules, which are displayed as a red surface, using HOLLOW [70]. (B) Structural alignment of DC-LAMP chain A (grey) and B (blue). Hydrogen bonds connect loop L1 to an adjacent hairpin in chain A, but not in chain B. (C) A V_H immunoglobulin domain is shown for comparison (PDB 1IGT). Complementarity determining regions (CDR) are drawn as red loops. The two β-sheets of the domain, displayed in green and blue, are connected by a disulfide bond (S-S).

Figure 6

Model of lysosomal membrane proteins and the glycoprotein coat. Structural models of glycosylated DC-LAMP and LAMP-1 were drawn to scale. The models are based on the DC-LAMP crystal structure and a hypothetical in silico model of LAMP-1. The thickness of the glycoprotein coat was reported to range from 5 to 12 nm with an average of 8 nm. The membrane-distal, N-terminal domain of LAMP-1 may adopt other positions. Dotted lines indicate putative binding sites consisting of the flexible loop L1 and an adjacent β-hairpin (Figure 5B). A box corresponding to 120 nm² surface area was drawn around a top view of LAMP-1. Polypeptides are depicted in green and glycans in yellow.

Figure 7

Molecular modeling. (A) The structure of the lowest energy obtained by molecular dynamics simulation is shown as a stick model and a van der Waals surface with pink carbon atoms. Glycans are shown with yellow carbon atoms. Prolines 203 and 206 have α-helical conformations. (B) Radius of gyration values during the simulation and r.m.s. deviations of the backbone atoms from the structure of lowest energy (marked in red). The peptide's lowest-energy structure had a radius of gyration of 14.6 Å, compared to 17.9 Å for the initial structure.