Structure of native glycolipoprotein filaments in honeybee royal jelly

Abstract

Royal jelly (RJ) is produced by honeybees (Apis mellifera) as nutrition during larval development. The high viscosity of RJ originates from high concentrations of long lipoprotein filaments that include the glycosylated major royal jelly protein 1 (MRJP1), the small protein apisimin and insect lipids. Using cryo-electron microscopy we reveal the architecture and the composition of RJ filaments, in which the MRJP1 forms the outer shell of the assembly, surrounding stacked apisimin tetramers harbouring tightly packed lipids in the centre. The structural data rationalize the pH-dependent disassembly of RJ filaments in the gut of the larvae.

Fig. 1. Architectural overview of the native RJ filaments.

a–d The native RJ helical assembly is shown as surface representation. a Side view, the helical rise is shown. b Cross section along the longitudinal axis. c As in a after 90° rotation along the longitudinal axis. d Top view, the helical rotation is shown. The MRJP1 subunits in each MRJP₄-apimisin₄ hetero-octamer layer are alternately colored in orange and cyan to distinguish subunits from different layers. Apisimin is colored in red, 24-methylenecholesterol in blue, and N-glycans in silver. e, f The H-shaped MRJP1₄/apisimin₄/24-methylenecholesterol₈ complex is shown from two orthogonal orientations with proteins represented as ribbons and the lipids as sticks. e Top view with the two 2-fold symmetry axes indicated as pink lines. f Side view along one of the 2-fold axes.

Fig. 2. The pH-dependent disassembly of RJ filaments.

a At pH 4.0, two major interfaces (labeled 1 and 2) between symmetry-related hetero-octamers exist in the RJ filament. Subunits (shown as surface in the overview and cartoons in the close-ups) are colored as in Fig. 1. The major contact (label 2) is mediated by homodimerization of MRJP1 alpha helices 1 (α1). b Top views of the electrostatic surface potentials (± 7 kT/e) of one hetero-octameric layer calculated at different pH values (see Methods for details). At pH 4.0, the four contact areas (1606 Ų in total) between the hetero-octamer layers (encircled in green) are predominantly hydrophobic and allow stable assembly of the hetero-octamers into RJ filaments. At the higher pH values observed in the gut of bee larvae, the interaction sites become negatively charged. The repulsion between the hetero-octamer layers leads to destabilization and eventually disassembly of the RJ filament. c Schematics showing the pH-dependent assembly model of RJ filaments.