Architecture of the native major royal jelly protein 1 oligomer

Abstract

Honeybee caste development is nutritionally regulated by royal jelly (RJ). Major royal jelly protein 1 (MRJP1), the most abundant glycoprotein among soluble royal jelly proteins, plays pivotal roles in honeybee nutrition and larvae development, and exhibits broad pharmacological activities in humans. However, its structure has long remained unknown. Herein, we identify and report a 16-molecule architecture of native MRJP1 oligomer containing four MRJP1, four apisimin, and eight unanticipated 24-methylenecholesterol molecules at 2.65 Å resolution. MRJP1 has a unique six-bladed β-propeller fold with three disulfide bonds, and it interacts with apisimin mainly by hydrophobic interaction. Every four 24-methylenecholesterol molecules are packaged by two MRJP1 and two apisimin molecules. This assembly dimerizes to form an H-shaped MRJP1₄-apisimin₄-24-methylenecholesterol₈ complex via apisimin in a conserved and pH-dependent fashion. Our findings offer a structural basis for understanding the pharmacological effects of MRJPs and 24-methylenecholesterol, and provide insights into their unique physiological roles in bees.

Fig. 1

Structure of the MRJP1 oligomer. a The H-like complex structure contains four MRJP1 (green or cyan), four apisimin (yellow or wheat), and eight 24-methylenecholesterol (blue, purple, orange, or lime sticks) molecules. N-acetylglucosamine (NAG) attached to Asn144 is shown in stick representation. The structure has two orthogonal twofold axes. Note that the stereoview is shown in the left and middle panels, a 90° rotation view in the right panel. The composite simulated-annealing mF_o—DF_c omit electron density maps of apisimin (b) and 24-methylenecholesterol (c) (also called Ostreasterol, Osl; formula shown in the right panel) are shown at 3.0 σ. The presence of apisimin (wheat arrow) was detected using tricine-SDS-PAGE

Fig. 2

Structural characterisation of the MRJP1 protomer. Secondary structures (a) and topology (b) of MRJP1 showing a six-bladed β-propeller fold. Three disulfide bonds and the central channel are shown

Fig. 3

Overlay of MRJP1 and apisimin with their closest structural homologs. Overlay of MRJP1 with Ava_4197 (PDB: 2qe8, a) and Ljm11 (PDB: 3q6k, b). c Overlay of the apisimin tetramer with the closest structural homolog, GrpE (PDB: 3a6m). Interactions between protomers are indicated by coloured arrows

Fig. 4

Detailed interactions of MRJP1 and apisimin in the MRJP1 dimer. a Overview of the three protein interfaces. Details of the symmetric intermolecular C-terminal and N-terminal interactions (b) and intermolecular C-terminal antiparallel β-sheets β31 (c), and their interactions (d) between MRJP1 protomers were calculated by Ligplot. Hemispheres represent hydrophobic interactions, and lines represent polar interactions. Residues from the two protomers are shown on the left and right, respectively. All residues involved in the hydrophobic interactions are shown in black. For hydrophilic interactions, residues are shown in red or blue. e Details of the interactions of apisimin

Fig. 5

Detailed interactions of Osl in the MRJP1 oligomer. a Four Osl molecules in the surface potential of MRJP1. b Interactions of the inner two Osl molecules with MRJP1 and apisimin. c Interactions of Osl2 calculated by Ligplot. d Interactions between the outer Osl4 and surrounding residues

Fig. 6

Structural basis of the formation of the MRJP1 oligomer. a The surface potential (±1 kBT/e) of the MRJP1 oligomer. The surface coloured as a gradient ranging from red (negative) to blue (positive). Details of the interfaces between apisimin and the neighbouring symmetry-generated apisimin molecule are shown in b. Two apisimin protomers are related by a twofold rotation axis, perpendicular to the plane of the page. c Analytical ultracentrifugation of the MRJP1 oligomer at pH 8. The c(S) distribution from sedimentation velocity analysis is shown. d Gel filtration of the MRJP1 oligomer at different pH values