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. 2017 Mar 21;56(11):1645-1655.
doi: 10.1021/acs.biochem.7b00020. Epub 2017 Mar 7.

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools

Samuel C Mandacaru  1   2 , Luis H F do Vale  2   3 , Siavash Vahidi  1 , Yiming Xiao  1 , Owen S Skinner  3 , Carlos A O Ricart  2 , Neil L Kelleher  3 , Marcelo Valle de Sousa  2 , Lars Konermann  1
Affiliations

Characterizing the Structure and Oligomerization of Major Royal Jelly Protein 1 (MRJP1) by Mass Spectrometry and Complementary Biophysical Tools

Samuel C Mandacaru et al. Biochemistry. .

Abstract

Royal jelly (RJ) triggers the development of female honeybee larvae into queens. This effect has been attributed to the presence of major royal jelly protein 1 (MRJP1) in RJ. MRJP1 isolated from royal jelly is tightly associated with apisimin, a 54-residue α-helical peptide that promotes the noncovalent assembly of MRJP1 into multimers. No high-resolution structural data are available for these complexes, and their binding stoichiometry remains uncertain. We examined MRJP1/apisimin using a range of biophysical techniques. We also investigated the behavior of deglycosylated samples, as well as samples with reduced apisimin content. Our mass spectrometry (MS) data demonstrate that the native complexes predominantly exist in a (MRJP14 apisimin4) stoichiometry. Hydrogen/deuterium exchange MS reveals that MRJP1 within these complexes is extensively disordered in the range of residues 20-265. Marginally stable secondary structure (likely antiparallel β-sheet) exists around residues 266-432. These weakly structured regions interchange with conformers that are extensively unfolded, giving rise to bimodal (EX1) isotope distributions. We propose that the native complexes have a "dimer of dimers" quaternary structure in which MRJP1 chains are bridged by apisimin. Specifically, our data suggest that apisimin acts as a linker that forms hydrophobic contacts involving the MRJP1 segment 316VLFFGLV322. Deglycosylation produces large soluble aggregates, highlighting the role of glycans as aggregation inhibitors. Samples with reduced apisimin content form dimeric complexes with a (MRJP12 apisimin1) stoichiometry. The information uncovered in this work will help pave the way toward a better understanding of the unique physiological role played by MRJP1 during queen differentiation.

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Figures

Figure 1
Figure 1
(a) Native ESI mass spectrum of the MRJP1/apisimin complex isolated from RJ (A+G+ sample). (b) Collisional activation of 30+ precursor ions releases highly charged MRJP monomers and apisimin. The peak marked with an asterisk corresponds to apisimin2+ with an unidentified 222.15 Da adduct. The inset in panel b shows apisimin ions in charge states of 1+ to 3+, generated by collisional activation of the intact complex. (c) Deconvoluted top-down fragmentation spectrum of MRJP112+ precursor ions. The inset in panel c shows the graphical fragment map, with cleavage sites corresponding to N-terminal (b-type) ions and C-terminal (y-type) ions that could be matched to the protein sequence with a mass accuracy of <5 ppm. The fragmentation map of apisimin is included in the inset of panel b.
Figure 2
Figure 2
BN-PAGE analyses of protein–protein interactions. Lane 1 contained MW markers. Lanes 2–5 show data for MRJP1 samples A+G+, AG+, A−G−, and A+G−, respectively.
Figure 3
Figure 3
(a) Far-UV CD spectra and (b) Trp fluorescence emission spectra of the four types of MRJP1 samples.
Figure 4
Figure 4
Kinetics of deuteration measured for various representative segments for the four types of MRJP1 samples. Each data point is the average of three independent measurements; error bars represent standard deviations.
Figure 5
Figure 5
Deuteration levels measured for MRJP1 peptides after HDX for 1 min. Data for the four different conditions are indicated in panels a–d. Panels on the right-hand side show difference plots relative to the A+G+ data: (e) A−G+ minus A+G+, (f) A−G− minus A+G+, and (g) A+G−minus A+G+.
Figure 6
Figure 6
Measured isotope distributions of MRJP1 segment 276–287 after deuteration for 10 s (a–d), and 100 min (e–h). The four columns of this figure are for the different types of samples, as indicated along the top. Also shown in each panel are Gaussian decompositions of the bimodal isotope distributions. Additional peptide data are shown in Figure S5.
Figure 7
Figure 7
Kyte–Doolittle hydropathy analyses of (a) MRJP1 and (b) apisimin, generated using http://web.expasy.org/protscale with a seven-residue averaging window and linear weighting. Key hydrophobic segments proposed to be involved in intermolecular contacts are highlighted.
Figure 8
Figure 8
Cartoon models of MRJP1/apisimin complexes. (a) A+G+ conditions favor (MRJP14 apisimin4) assemblies; apisimin acts as a hydrophobic linker that connects the relatively structured MRJP1 C-terminal regions. The N-terminal regions are disordered. Apisimin-depleted samples (b) A−G+ and (c) A−G− form (MRJP12 apisimin1) complexes. (d) A+G− samples undergo apisimin-mediated formation of soluble aggregates. MRJP1 is colored light blue. Apisimin is colored dark purple. Glycans at N144 and N177 are colored red. The hydrophobic MRJP1 segment of residues 316–322 is colored yellow.
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